DETECTING TARGETS USING MASS TAGS AND MASS SPECTROMETRY
Particular disclosed embodiments disclosed herein concern using a one or more various mass tags, which can be specifically deposited at targets through direct or indirect enzymatic-catalyzed transformation, to provide a method for identifying targets in tissue samples. The mass tags may be labeled with stable isotopes to produce mass tags having the same chemical structure but different masses. Mass codes produced by ionizing the mass tags are detected and/or quantified using mass spectrometry. The method can be used for multiplexed detection of multiple targets in a particular sample. In some embodiments, a map divided into sections representing sections of the tissue sample may be prepared, with the map sections including data corresponding to quantification data wherein the size of a mass peak is determined and correlated with the amount of a target for the corresponding tissue sample section.
The present application is a continuation of U.S. patent application Ser. No. 14/981,181, filed on Dec. 28, 2015, which is a continuation of U.S. patent application Ser. No. 13/805,983, filed Jan. 25, 2013, which is the National Stage of International Application No. PCT/US2011/042853, filed Jul. 1, 2011, which claims the benefit of the earlier filing date under 35 U.S.C. § 119 (e) of U.S. provisional patent application No. 61/398,944, filed Jul. 2, 2010, U.S. provisional patent application No. 61/398,942, filed Jul. 2, 2010, U.S. provisional patent application No. 61/464,937, filed Mar. 11, 2011, and U.S. provisional patent application No. 61/464,977, filed Mar. 11, 2011, the disclosures of which are hereby incorporated by reference herein in their entirty.
FIELDThe present disclosure concerns embodiments of compounds, compositions comprising such compounds, and methods for using such compounds and compositions for imaging a target and/or multiple targets in a sample, and kits for performing the methods. In particular, the present disclosure concerns using enzymatic reactions to localize mass tags proximal to or at one or more targets in a tissue sample, and detecting mass codes enzymatically deposited at the target(s) using mass spectrophotometric techniques.
BACKGROUNDCancer is one of the most common diseases worldwide. Biomarkers play a key role in cancer diagnosis and prognosis. Determining the presence of and/or quantities (relative amounts and/or absolute amounts) of multiple biomarkers are useful for accurate prediction and prognosis of cancers, and survival rate may depend at least in part on targeted therapy based upon accurate detection and quantification, wherein quantification comprises determining the size of a mass peak and correlating it with the amount of particular biomarkers. For example, in breast cancer, the relative abundances of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2) biomarkers correlates to the patient's chances of surviving five or more years.
Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify cancer patients. Conventional IHC methods using optical imaging are suitable for detecting one or more targets within a sample (e.g., a tissue sample). However, IHC methods cannot provide accurate quantitative results for the detected targets. Typically, only certified medical personnel are sufficiently skilled to evaluate a subject's diagnosis and prognosis based upon IHC optical imaging results. Currently, there is no suitable IHC platform for quantitative multiplexed assays useful for assessing cancers and determining personalized cancer therapy.
A number of methods have been developed for detecting and imaging biomolecules, such as nucleic acids and proteins. Nucleic acids, for example, may be analyzed using labeled probe molecules. The labels are detected to determine whether specific binding or hybridization has taken place. Various probe labeling methods are known, including radioactive atoms, fluorescent dyes, luminescent reagents, electron capture reagents and light absorbing dyes. Each of these methods has features suitable for certain applications, but there also are inherent limitations to each such method.
Mass spectrometry has been increasingly used for bioanalytical analyses. Mass spectrometry his well suited for multiplexing because mass differentiation allows many simultaneous detection channels. However, complex biomolecules, such as DNA, have complex mass spectra and may be difficult to detect in a matrix due to relatively poor sensitivity.
SUMMARYA need exists in the art for additional tissue analysis and imaging techniques. Certain embodiments address many of the drawbacks associated with prior techniques, and allows for the use of more durable tissue samples, rather than fresh, frozen tissue samples used in other techniques. Matrix-assisted mass spectrometry may not be required with the disclosed method, which allows sample detection without the background interference, and eliminates “hot spots” that may result from using a matrix, crystal formation, or laser power limitations associated with the use of MALDI mass spectrometry. Other embodiments also allow substantial signal amplification, which provides much more sensitive detection and improved spatial resolution than techniques that do not involve signal amplification. All methods result in substantial signal amplification, allowing detection and imaging for both protein and nucleic acid.
A need also exists for a method to quantify, particularly in a single sample, multiple targets, e.g., cancer biomarkers, on formalin-fixed, paraffin-embedded (FFPE) tissue for diagnosis and prognosis. Certain disclosed embodiments address these and other needs.
Disclosed herein are embodiments of a conjugate, comprising a mass tag precursor, an enzyme substrate or a tyramine moiety or a tyramine derivative, and an optional linker. In particular disclosed embodiments, the mass tag precursor may be isotopically labeled. Also disclosed are embodiments of a method for detecting a target in a sample, and embodiments of a kit comprising the disclosed mass tag precursors.
Other embodiments utilize several classes of mass tag precursors including, by way of example and without limitation, triarylmethane derivatives, such as substituted triarylmethane compounds, triarylmethane dyes, triarylmethane-diazonium species, azo-naphthol carbonyl compounds, ultra small metal clusters, oligomers of peroxidase (e.g. horseradish peroxidase) substrates, such as aniline and phenol derivatives, certain cationic dyes, nitrophenyl compounds, heteraryl compounds, metal-heteraryl complexes, nanoparticle-based compounds, and any isotopically-labeled derivative thereof.
Other embodiments of the disclosed mass tag precursors concern azo dyes that are formed by the addition of various phenol, aniline, or naphthol derivatives, which are generated as a product of an enzymatic reaction (e.g. cleavage using a phosphatase, or glycosidase catalyzed hydrolysis), with various diazonium salts. The naphthol/azo dye is usually of deep color and specifically precipitates at the target. Laser-mediated cation formation can be facilitated by the presence of electron donating groups in the aromatic conjugated systems of the naphthol/azo mass tag.
Another class of mass tag precursors that are disclosed includes various substrates for a peroxidase (e.g. horseradish peroxidase [HRP]), including substituted aryl and heteroaryl compounds. This class of mass tag precursors encompasses substituted aryl and heteroaryl compounds that are capable of oligomerization, such as diaminobenzidine, diaminonaphthalenes, and aminoquinolines. Tissue targets labeled with a peroxidase, such as HRP, can be treated with these compounds and LDI mass spectrometry can be used to ionize the oligomeric mass tags formed through enzymatic reactions, followed by detecting the mass codes.
Still other disclosed embodiments concern heteroaryl mass tag precursors that undergo fragmentation upon exposure to an energy source. Disclosed embodiments may comprise azine moieties (having only one nitrogen atom in the aromatic ring), diazine moieties (having two nitrogen atoms in the aromatic ring), triazine moieties (having three nitrogen atoms in the aromatic ring), and tetrazine (having four nitrogen atoms in the aromatic ring) moieties. Particular disclosed embodiments utilize a tetrazine mass tag precursor, which readily undergoes fragmentation upon exposure to an energy source.
Naphthol derivatives can be used with cationic dyes to comprise another class of effective mass tag precursors. In a particular embodiment, naphthol derivatives precipitate at the desired site by catalyzed oxidation and can be further stained with various cationic dyes. These dyes are mostly cationic and have high absorbance at the wavelength of the laser used in LDI mass spectrometry, and are therefore useful candidates for mass tag precursors.
Another class of mass tag precursors concern nitrophenyl moieties, which undergo photolysis to release a corresponding mass code. In certain disclosed embodiments, the mass code comprises a linker (such as a polyethylene glycol linker), a charged moiety (such as arginine, lysine, histidine, or phosphate), and/or a chromophore (such as a hapten or dye).
In some disclosed embodiments, a metal-heteroaryl mass tag precursor may be used. Embodiments of these mass tag precursors can be selected from metal-monoheteroaryl complexes, metal-biheteroaryl complexes, and metal-terheteroaryl complexes. More typically these metal-heteroaryl mass tag precursors are selected from metal-monopyridine complexes, metal-bipyridine complexes, and metal-terpyridine complexes. Other metal-complexed mass tag precursors are disclosed, wherein the mass tag precursor is a metal-peptide complex. These complexes can be ionized to form detectable mass codes. In particular embodiments, these mass tag precursors comprise a nickel complex which is bound to two or more equivalent or different amino acids. Exemplary amino acids include any amino acid (e.g. histidine) capable of chelating with a metal.
In some disclosed embodiments, a nanoparticle functionalized with mass tag ligands may be used as a mass tag precursor. The mass tag ligands are bound to the nanoparticle via a binding moiety. The mass tag-functionalized nanoparticles are also coupled to one or more tyramine moieties and/or tyramine derivatives. In other disclosed embodiments, the conjugates include the nanoparticle, itself, as a mass tag precursor, which produces small cluster metal ions upon exposure to an energy source. These small cluster metal ions act as a mass code. These disclosed embodiments use metals or a semiconductor, wherein the metals can be selected from Groups 3-15 of the periodic table. Typically, metals are selected from Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, Bi, or a combination thereof.
Illustrative embodiments include mass tag precursors conjugated to enzyme substrate moieties, such as phosphates, galactosides, and lactams. These disclosed embodiments of the disclosed conjugate typically are water soluble, but are deposited on a desired target by enzymatic cleavage to form water insoluble mass tags.
In certain disclosed embodiments, the mass tag precursor is coupled to a tyramine or tyramine derivative via a linker, such as an aliphatic, heteroaliphatic, or a heterobifunctional linker. Particular disclosed embodiments concern heteroaliphatic linkers, such as polyethylene glycol units having a formula PEGn where n ranges from 1 to about 50, 1 to about 30, 1 to about 25, 1 to about 10, and 4 or 8. Other embodiments use heterobifunctional linkers to form mass tag precursor—tyramine conjugates or mass tag precursor-tyramine derivative conjugates.
In some embodiments, multiple mass tag precursors, tyramine moieties, and/or tyramine derivatives are conjugated to multivalent carriers. These carriers include polymers, biomolecules, liposomes, micelles, and nanoparticles. Polymers can be linear (such as a polyacid, polyamine, polysaccharide, polyhydrazine, or co-polymer) or hyperbranched (such as polyethyleneimine or dendrimers). A biomolecule carrier can also be used, and may be selected from a protein, a polypeptide, an oligopeptide, a peptide, a nucleic acid, DNA, RNA, an oligosaccharide, a polysaccharide, and monomers thereof. Nanoparticles are comprised of a semiconductor, metal or multiple metals, selected from Group 3-15 of the periodic table. Particular embodiments utilize nanoparticles comprising Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, and Bi, or a combination thereof.
Particular disclosed embodiments of the disclosed conjugate can be formed using any of the disclosed mass tag precursors, optional linkers or carriers, tyramine moieties, or tyramine derivatives. Particular disclosed embodiments of the disclosed conjugate comprising a triarylmethane mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier; a nitrophenyl mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier; a heteroaryl mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier; a naphthol-azo mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier; a metal-heteroaryl chelate mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier; a ligand mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier; and a nanoparticle as a mass tag precursor in conjunction with a tyramine or tyramine derivative, either conjugated directly or through a linker or carrier. Any of these disclosed embodiments can be used in disclosed embodiments of the disclosed method. These disclosed embodiments can also be utilized for multiplexing in order to detect multiple targets in a sample.
Also contemplated in the disclosed embodiments of the disclosed conjugate are isotopically-labeled derivatives of any of the disclosed mass tag precursors.
A method for detecting a target in a sample concerns contacting a sample with an enzyme-specific binding moiety conjugate selected to recognize the target. The sample then is contacted with a mass tag precursor conjugate, comprising a mass tag precursor and an enzyme substrate, a tyramine moiety, or a tyramine derivative, and an optional linker. The mass tag precursor conjugate undergoes reaction with the enzyme or with the product of the enzymatic reaction to produce precipitated mass tags, covalently bound mass tags, or non-covalently bound mass tags. The sample is exposed to an energy source, which provides sufficient energy to produce a mass code from the mass tag. After ionization, the mass code can be detected using a detection method, such as mass spectrometry. In some embodiments, the sample is exposed to a first solution comprising the enzyme-specific binding moiety conjugate and a second solution comprising the mass tag precursor conjugate. Enzyme moieties of the enzyme-specific binding moiety can be selected from oxido-reductase enzymes (e.g. peroxidases), phosphatases (e.g. alkaline phosphatase), lactamases (e.g. β-lactamase), and galactosidases (e.g. β-D-galactosidase, β-galactosidase). Specific binding moieties can be selected from a protein, a polypeptide, an oligopeptide, a peptide, a nucleic acid, DNA, RNA, an oligosaccharide, a polysaccharide, and monomers thereof. Particular disclosed embodiments concern using alkaline phosphatase-antibody conjugates and horseradish peroxidase-antibody conjugates. In some disclosed embodiments, a specific binding moiety recognizes the target. In other disclosed embodiments, the specific binding moiety recognizes a primary antibody bound to the target.
In addition, certain disclosed embodiments utilize an active species, generated as the product of an enzymatic reaction, to deposit a mass tag at the target. The reactions between the active species and the mass tag precursor include, but are not limited to, redox reactions, addition reactions, elimination reactions and substitution reactions. For example, metal ions can be reduced in the presence of various reductants, such as hydroquinone, in the presence of hydrogen peroxide and horseradish peroxidase. This redox process causes the reduced metal to precipitate at a particular target site, followed by subsequent detection of the metal cluster mass codes using LDI-TOF mass spectrometry.
Certain disclosed embodiments concern a method for imaging and quantifying multiple targets in a sample, such as a tissue sample, using stable-isotope-labeled mass tag precursors and mass spectrometric (MS) techniques, such as laser desorption/ionization, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The targets may be proteins, peptides, nucleic acid sequences, or any combination thereof. In some embodiments, the targets are cancer biomarkers, and mass tag precursors are used to identify and/or quantify each target. For example, the cancer biomarkers may be breast cancer biomarkers, such as ER, PR, Her2, and/or Ki67. In particular embodiments, the targets are different forms of the same cancer biomarker, e.g., intact and truncated forms, such as intact and truncated forms of Her2. In certain embodiments, the sample is formalin-fixed, paraffin-embedded (FFPE) tissue. The mass tag precursors used to identify and/or quantify multiple targets within a single tissue sample are labeled with stable isotopes (e.g., 2H, 13C, 15N, etc.) to produce mass tag precursors having the same chemical structure but different masses. In particular embodiments, the mass tag precursors are stable-isotope-labeled chromogens useful for IHC tissue staining.
One advantage of the disclosed method is that it can be used for multiplexed detection of multiple targets in a particular sample. For example, consecutive enzymatic reactions can be used to distribute several mass tags at several different target locations in a sample. Many embodiments provide new methods for tissue analysis through the use of a wide class of mass tag precursors that provide effective and efficient methods for detection of particular target sites. The mass tag precursors, mass tags, mass codes, and method of using the mass tag precursors conjugates described in the current method are intended for the use in tissue mass imaging applications, especially for FFPE tissue samples. However, certain embodiments can also be utilized for enzyme-linked immunoassays in general. The adoption of these methods in enzyme-linked immunoassays offers enhanced sensitivity and quantifiability in a multiplexing fashion.
In some embodiments, the method comprises depositing a first mass tag at a first target in a tissue sample and depositing a second mass tag at a second target in the tissue sample, ionizing each mass tag to produce a mass code corresponding to a target at which the mass tag was deposited, detecting each mass code, and quantifying each target by quantifying an amount of the mass code deposited at each target. The first mass tag and the second mass tag have the same chemical structure, but at least one of the first mass tag and the second mass tag is isotopically labeled such that they differ in mass, thereby producing respective mass codes having the same chemical structure but differing in mass. In certain embodiments, the method further includes depositing additional mass tags at one or more subsequent targets in the tissue sample, wherein each additional mass tag has the same chemical structure as the first and second mass tags and is isotopically labeled such that its mass differs from any other mass tag used to identify and quantify any other target in the tissue sample. In particular embodiments, detecting each mass code is performed using mass spectrometry, and each mass code corresponding to a target differs in mass from any other mass code corresponding to any other target by a sufficient mass amount to prevent overlap of mass spectrometric peaks from two or more mass codes.
In some embodiments, an amount of a mass code is quantified by measuring a mass spectrometric peak having a m/z ratio corresponding to an expected m/z ratio of the mass code. In other embodiments, a plurality of mass spectrometric peaks is measured and the measurements are combined to quantify the amount of the mass code. The plurality of peaks includes a primary peak having a m/z ratio of x corresponding to the expected m/z ratio of the mass code and at least one secondary peak having a m/z ratio of x+n where n is an integer ≥1 and x+n is less than a m/z ratio corresponding to an expected m/z ratio of any other mass code present.
In some embodiments, depositing a mass tag includes immobilizing an enzyme at a target, and contacting the tissue sample with an enzyme substrate moiety and a mass tag precursor. The enzyme substrate moiety reacts with the enzyme and the mass tag precursor to produce and deposit a mass tag at the target. When two or more targets are present in the tissue sample, mass tags are deposited sequentially at each target as described above. After a mass tag is deposited, the corresponding enzyme is deactivated prior to depositing a subsequent mass tag at a subsequent target. In other disclosed embodiments, the enzyme reacts with a mass tag precursor-tyramine conjugate or a mass tag precursor—tyramine derivative conjugate to deposit, typically covalently, the mass tag proximal to the target.
In some embodiments, immobilizing an enzyme at a target includes contacting the tissue sample with a conjugate comprising a specific binding moiety and an enzyme. In certain embodiments, the specific binding moiety is an antibody.
The specific binding moiety is capable of recognizing and binding directly to the target or to another specific binding moiety previously bound to the target. In particular embodiments, the first enzyme, the second enzyme, and any additional enzyme are the same.
In some embodiments, the target is a protein or a peptide, and the specific binding moiety previously bound to the target is an antibody. In some embodiments, the target is a nucleic acid sequence, and the specific binding moiety previously bound to the target is a labeled probe capable of hybridizing to the nucleic acid sequence or a portion thereof.
In some embodiments, the mass tags are ionized by exposing one or more mass tags to laser-initiated ionization/desorption to produce a mass code. The one or more mass codes are detected and quantified. The first target is quantified by quantifying the first mass code, and the second target is quantified by quantifying the second mass code. In certain embodiments, the laser ionizes mass tags in a section of the tissue sample. In particular embodiments, the tissue sample and/or laser subsequently is moved such that the laser is positioned to ionize mass tags in a subsequent section of the tissue sample, and mass codes from the subsequent section are detected and quantified. The steps of moving the sample and the laser relative to one another, ionizing mass tags, and detecting and quantifying the mass codes can be performed in a plurality of subsequent sections of the tissue sample.
In some embodiments, a representative map of the sample is constructed. Certain disclosed embodiments concern a map that includes a plurality of sections corresponding to the tissue sample sections, and each map section includes data correlating to mass code quantification wherein quantification comprises determining the size of a mass peak and correlating it with the amount the target for the corresponding tissue sample section. In certain embodiments, the data is presented in representative form, whereby a data representative such as a color, symbol, etc. is provided that corresponds to a numerical value for the data correlating to the mass code quantification. In particular embodiments, the data correlates to a ratio of two mass codes quantified for two targets in the corresponding tissue sample section.
In some embodiments, first and second mass tags are deposited at first and second targets, and the diazonium salts used to react with a naphthol moiety, thus forming a first mass tag precursor and second mass tag precursor, have the structures
where n is 1, 2, 3, 4, or 5. Preferably n is 2-5, with disclosed working examples having n=5 as shown below.
In certain embodiments, the tissue sample includes two to four targets, and the diazonium salts selected to ultimately produce the first mass tag precursor, the second mass tag precursor, and any additional mass tag precursors are selected from
where n is 0-5, and R1 and R1″ are independently lower alkyl optionally substituted with one or more isotopes, e.g., —CD3, —CD2CD3, etc. In some examples, the diazonium salts are selected from
Embodiments of a kit for detecting plural targets by mass spectrometry include at least a first mass tag precursor and a second mass tag precursor and at least one conjugate comprising an enzyme and a specific binding moiety capable of recognizing and binding to a target in a tissue sample or to another specific binding moiety previously bound to a target in a tissue sample. The first and second mass tag precursors have the same chemical structure, except that at least one of the precursors is isotopically labeled such that the first and second mass tag precursors differ in mass from one another.
In some embodiments, the kit also includes an enzyme substrate moiety capable of reacting with the enzyme and the first mass tag precursor to produce and deposit a first mass tag at a first target in the tissue sample. The enzyme substrate moiety further is capable of reacting with the enzyme and the second mass tag precursor to produce and deposit a second mass tag at a second target in the tissue sample.
In certain embodiments, the kit includes one or more additional mass tag precursors, wherein each additional mass tag precursor has the same chemical structure as the first and second mass tag precursors, except that each additional mass tag precursor is isotopically labeled such that each additional mass tag precursor differs in mass from the mass of the first mass tag precursor, the second mass tag precursor, and any other additional mass tag precursor. In such embodiments, the enzyme substrate moiety is capable of reacting with the enzyme and each additional mass tag precursor to produce and deposit an additional mass tag at an additional target in the tissue sample.
In some embodiments, the kits include one or more mass tag precursor conjugates, comprising a mass tag precursor, an enzyme substrate, or a tyramine or a tyramine derivative, an optional linker, and an optional carrier. Each conjugate is capable of reacting with the enzyme to deposit a mass tag at a particular target tin the tissue sample.
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The nucleic and amino acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The sequence listing is submitted as an ASCII text file, named “87482-01_ST25,” created on Dec. 17, 2015, 97KB, which is incorporated by reference herein.
SEQ ID NO: 1 is a Her1 amino acid sequence (NCBI Reference Sequence No. NP_005219.2).
SEQ ID NO: 2 is an exemplary cDNA sequence encoding Her1 (NCBI Reference Sequence No. NM_005228.3).
SEQ ID NO: 3 is a Her2 amino acid sequence (NCBI Reference Sequence No. NP_004439.2).
SEQ ID NO: 4 is an exemplary cDNA sequence encoding Her2 (NCBI Reference Sequence No. NM_004448.2).
SEQ ID NO: 5 is a Her3 amino acid sequence (NCBI Reference Sequence No. NP_001973.2).
SEQ ID NO: 6 is an exemplary cDNA sequence encoding Her3 (NCBI Reference Sequence No. NM_001982.3).
SEQ ID NO: 7 is a Her4 amino acid sequence (Genbank Accession No. AAI43750).
SEQ ID NO: 8 is an exemplary cDNA sequence encoding Her4 (GenBank Accession No. BC143749.1).
SEQ ID NO: 9 is an Estrogen Receptor amino acid sequence (NCBI Reference Sequence No. NP_000116.2).
SEQ ID NO: 10 is an exemplary cDNA sequence encoding an Estrogen Receptor (NCBI Reference Sequence No. NM_000125.3).
SEQ ID NO: 11 is a Progesterone Receptor amino acid sequence (Genbank Accession No. AAD01587.1).
SEQ ID NO: 12 is an exemplary cDNA sequence encoding a Progesterone Receptor (GenBank Accession No. AF016381.1).
DETAILED DESCRIPTION I. TERMS AND ABBREVIATIONSUnless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.
As used herein, the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Also, as used herein, the term “comprises” means “includes.” Hence “comprising A or B” means including A, B, or A and B. It is further to be understood that all nucleotide sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides or other compounds are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All sequences referred to by GenBank Accession numbers herein are incorporated by reference as they appeared in the database on Jun. 30, 2011.
In order to facilitate review of the various examples of this disclosure, the following explanations of specific terms and abbreviations are provided:
Aliphatic: Any open or closed chain molecule, excluding aromatic compounds, containing only carbon and hydrogen atoms which are joined by single bonds (alkanes), double bonds (alkenes), or triple bonds (alkynes). This term encompasses saturated and unsaturated aliphatic compounds.
Amplification: Certain embodiments of the present invention allow a single target to be detected using plural visualization complexes, where the complexes can be the same or different, to facilitate identification and/or quantification wherein quantification comprises determining the size of a mass peak and correlating it with the amount of a particular target.
Analog or Derivative: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure or mass, such as a difference in the length of an alkyl chain or the inclusion of one of more isotopes), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure.
Antibody: “Antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules (including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant, or binding affinity, for the molecule of interest that is at least 103 M−1 greater, at least 104 M−1 greater or at least 105 M−1 greater than a binding constant for other molecules in a biological sample. In one embodiment, binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay.
More particularly, “antibody” refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
Antigen: A compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids, proteins, and peptides. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
Aromatic: A term describing conjugated rings having unsaturated bonds, lone pairs, or empty orbitals, which exhibit a stabilization stronger than would be expected by the stabilization of conjugation alone. It can also be considered a manifestation of cyclic delocalization and of resonance.
Aryl: A substantially hydrocarbon-based aromatic compound, or a radical thereof (e.g. C6H5) as a substituent bonded to another group, particularly other organic groups, having a ring structure as exemplified by benzene, naphthalene, phenanthrene, anthracene, etc.
Aryl alkyl: A compound, or a radical thereof (C7H7 for toluene) as a substituent bonded to another group, particularly other organic groups, containing both aliphatic and aromatic structures.
Biomarker: A biological marker or indicator of a biological state that may be measured or evaluated to indicate a particular disease state or other physiological state of an organism. Biomarkers can indicate a change in expression or state of a protein that correlates with a disease risk or progression. Biomarkers can be used to measure the progression of a disease. Biomarkers also can be used to predict or measure the efficacy of a particular treatment for a disease. Biomarkers can be, for example, specific cells, molecules, genes, gene products, enzymes, or hormones.
Carrier: A molecule to which another molecule can be bound, such as a hapten, a mass tag, a mass tag precursor, or an antigen. Carrier molecules include immunogenic carriers and specific-binding carriers. When bound to an immunogenic carrier, the bound molecule may become immunogenic. Immunogenic carriers may be chosen to increase the immunogenicity of the bound molecule and/or to elicit antibodies against the carrier, which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T-cell dependence. Useful carriers include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Specific binding carriers can be any type of specific binding moiety, including an antibody, a nucleic acid, an avidin, a protein-nucleic acid. The carrier can be water soluble or insoluble, and in some embodiments is a protein or polypeptide.
Chromogen: An aromatic compound comprising a chemical grouping, or chromophore, that gives color to the compound by causing displacement of, or appearance of, absorbent bands in the visible spectrum. Exemplary chromophores include, but are not limited to, —NO (e.g., nitroso dyes), —NO2 (e.g., nitro dyes), and —N═N— (e.g., azo dyes).
Conjugate: A molecule comprising two independent molecules, which have been joined through a bond (typically a covalent or ionic bond), such as a mass tag precursor joined with an enzyme substrate.
Conjugating, joining, bonding or linking: Covalently linking one molecule to another molecule to make a larger molecule. For example, making two polypeptides into one contiguous polypeptide molecule, or covalently attaching a mass tag precursor, hapten or other molecule to a polypeptide, such as an scFv antibody.
Coupled: A first atom and/or molecule being attached to a second atom and/or molecule. Coupling generally refers to attaching the atoms or molecules by non-covalent means. The term “coupled” refers to both directly coupled and indirectly coupled. A secondary antibody provides an example of indirect coupling. One specific example of indirect coupling is a rabbit anti-hapten primary antibody that is bound by a mouse anti-rabbit IgG antibody, that is in turn bound by a goat anti-mouse IgG antibody that is covalently linked to a detectable label; thus, the detectable label is “coupled” indirectly to the mouse anti-rabbit IgG antibody and to the rabbit anti-hapten primary antibody.
Deactivat(e/ion): Deactivate and deactivation generally refer to inhibiting an enzyme or making an enzyme inactive. Examples of deactivating and deactivation include the following: mechanically removing an enzyme after it has reacted with the enzyme substrate-mass tag conjugate, such as by washing away the enzyme via elution of the secondary antibody, to which the enzyme is conjugated, as well as the primary antibody (if present); chemically deactivating the enzyme after it has reacted with the enzyme substrate-mass tag conjugate, such as by exposing the sample comprising the enzyme to an oxidant, such as hydrogen peroxide; and/or chemically inhibiting the enzyme after it has reacted with the enzyme substrate-mass tag conjugate with an agent capable of inhibiting a particular enzymatic action. Deactivation encompasses any of these techniques, any combination thereof, and any other technique now known or hereafter developed that achieves the stated purpose.
Detectable Label: A detectable compound or composition that is attached directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymes, and radioactive isotopes.
Derivative: In chemistry, a derivative is a compound that is derived from a similar compound or a compound that can be imagined to arise from another compound, for example, if one atom is replaced with another atom or group of atoms. The latter definition is common in organic chemistry. In biochemistry, derivative refers to compounds that at least theoretically can be formed from the precursor compound.
Effective amount: As used herein, an effective amount is an amount sufficient to produce a desired effect, e.g., an amount sufficient to enable detection of a target or analyte.
Epitope: An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope.
ER: Estrogen receptor.
FFPE: Formalin-fixed, paraffin-embedded.
Hapten: A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule.
Human epidermal growth factor receptor (Her) family: A family of structurally related proteins, including at least Her1, Her2, Her3 and Her4 (aka EGFR1, EGFR2, EGFR3 and EGFR4, respectively, or ErbB-1, ErbB-2, ErbB-3 and ErbB-4, respectively). Her1, Her2 and Her4 are receptor tyrosine kinases; although Her3 shares homology with Her1, Her2 and Her4, Her3 is kinase inactive. Included in the Her family is p95, a truncated form of Her2 lacking portions of the Her2 extracellular domain (see, e.g., Arribas et al., Cancer Res., 71:1515-1519, 2011; Molina et al., Cancer Res., 61:4744-4749, 2001).
The human epidermal growth factor family of receptors mediate cell growth and are disregulated in many types of cancer. For example Her1 and Her2 are upregulated in many human cancers, and their excessive signaling may be critical factors in the development and malignancy of these tumors. See, e.g., Herbst, Int. J. Radiat. Oncol. Biol. Phys., 59:21-6, 2004; Zhang et al., J. Clin. Invest. 117 (8): 2051-8, 2007. Receptor dimerization is essential for Her pathway activation leading to receptor phosphorylation and downstream signal transduction. Unlike Her1, −3 and −4, Her2 has no known ligand and assumes an open conformation, with its dimerization domain exposed for interaction with other ligand-activated Her receptors.
Approximately 30% of breast cancers have an amplification of the Her2 gene or overexpression of its protein product. Her2 overexpression also occurs in other cancer types, such as ovarian cancer, stomach cancer, and biologically aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. See, e.g., Santin et al., Int. J. Gynaecol. Obstet., 102 (2): 128-31, 2008. Her2-containing homo- and hetero-dimers are transformation competent protein complexes. Trastuzumab, a humanized antibody that prevents Her2 homodimerization is used to treat certain Her2 overexpressing cancers, including breast cancer. Additionally, the level of Her2 expression in cancer tissue is predictive of patient response to Her2 therapeutic antibodies (e.g., Trastuzumab). Because of its prognostic role as well as its ability to predict response to Trastuzumab, tumors (e.g., tumors associated with breast cancer) are routinely checked for overexpression of Her2.
The Her pathway is also involved in ovarian cancer pathogenesis. Many ovarian tumor samples express all members of the Her family. Co-expression of Her1 and Her2 is seen more frequently in ovarian cancer than in normal ovarian epithelium, and overexpression of both receptors correlates with poor prognosis. Preferred dimerization with Her2 (Her1/Her2, Her2/Her3) and subsequent pathway activation via receptor phosphorylation have also been shown to drive ovarian tumor cell proliferation, even in the absence of Her2 overexpression. Pertuzumab, a humanized antibody that prevents Her2 dimerization (with itself and with Her3) has been shown to provide therapeutic benefit to patients with Her2 and/orHer3 expressing ovarian cancer.
Examples of Her1 amino acid sequence include NCBI/Genbank accession Nos. NP_005219.2, CAA25240.1, AAT52212.1, AAZ66620.1, BAF83041.1, BAH11869.1, ADZ75461.1, ADL28125.1, BAD92679.1, AAH94761.1. Examples of, Her2, amino acid sequences include NCBI/Genbank accession BAJ17684.1, P04626.1, AAI67147.1, NP_001005862.1, NP_004439.2, AAA75493.1, AAO18082.1. Examples of Her3 amino acid sequences include NCBI/Genbank accession Nos. NP_001973.3, P21860.1, AAH82992.1, AAH02706.1, AAA35979.1. Examples of Her4 amino acid sequences include NCBI/Genbank accession Nos., AAI43750, Q15303.1, NP_005226.1, NP_001036064.1, AAI43748.1.
Heteroaliphatic: An aliphatic group, which contains one or more atoms other than carbon and hydrogen, such as, but not limited to, oxygen, sulfur, nitrogen, phosphorus, chlorine, fluorine, bromine, iodine, and selenium.
Heterobifunctional: A molecule or group of atoms that contains at least two different reactive groups (typically one reactive group at each end), which are reactive towards particular functional groups, including but not limited to sulfhydryls and amines. For example, a heterobifunctional molecule can be used to create chemical covalent bonds between two or more molecules.
IHC: Immunohistochemistry.
Ionization: The process of converting an atom or molecule into an ion by adding or removing charged particles such as electrons, protons, or other polyatomic ions.
Immunogenic Conjugate or Composition: A composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a vertebrate. In some embodiments, the immunogenic response is protective or provides protective immunity by enabling the vertebrate animal to better resist infection or disease progression from the organism against which the immunogenic composition is directed. One specific example of a type of immunogenic composition is a vaccine.
Immunogen: A compound, composition, or substance which is capable, under appropriate conditions, of stimulating the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal.
Immunologically reactive conditions: Includes reference to conditions which allow an antibody raised against a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions. The immunologically reactive conditions employed in the methods are “physiological conditions” which include reference to conditions (such as temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0° C. and below 50° C. Osmolarity is within the range that is supportive of cell viability and proliferation.
Isolated: An “isolated” component has been substantially separated or purified from other components, e.g., other components of a solution, a mixture, or a cell. An “isolated” biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins, or fragments thereof.
Isotope: One of two or more forms of an element that have the same atomic number, but different masses. The mass differences are due to the presence of one or more extra neutrons in the nucleus. For example, deuterium and tritium are isotopes of hydrogen. Hydrogen has one proton, one electron, and no neutrons, and has a mass of 1 dalton. Deuterium has one proton, one electron, and one neutron, and has a mass of 2 daltons. Tritium has one proton, one electron, and two neutrons, and has a mass of 3 daltons.
Linker: As used herein, a linker is a molecule or group of atoms positioned between two moieties. For example, a mass tag precursor conjugate may include a linker between the mass tag and the enzyme substrate moiety. Typically, linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two moieties. The two functional groups may be the same, i.e., a homobifunctional linker, or different, i.e., a heterobifunctional linker.
Linker peptide: A peptide positioned between two moieties. One example of a linker peptide is an antibody binding fragment (such as an Fv fragment) that indirectly bonds the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as a scFv, to an effector molecule, such as a cytotoxin or a detectable label.
Lower alkyl: Any aliphatic chain that contains 1-10 carbon atoms.
Mass Code/Reporter Ion: An ion that is produced from a mass tag during an ionization step that can be detected, such as by a mass spectrometer. For example, a photolabile moiety, having at least one photolabile bond may be desorbed and ionized to form a mass code. The mass code, or reporter ion, can be an ionized form of the mass tag (e.g. soft ionization) or can be an ionized fragment.
Mass Tag: A moiety that is capable of producing a mass code upon ionization. Mass tag moieties may comprise one or more labile bonds, such as photolabile bonds, that will cleave, ionize, or dissociate upon photolysis. In some embodiments, the mass tag is equivalent to a mass code, such as when the mass tag comprises a charged moiety. Mass tags may be selected according to the properties that make them useful for ionization and/or the properties that make them useful for deposition proximal to the enzyme. Stability of the mass tag or its dominant fragments as ions, susceptibility to laser ablation, and dissociation from the matrix may all distinguish mass tags from other compounds.
Mass tag precursor: A moiety that is capable of undergoing a reaction to form a mass tag. For example, a mass tag precursor may be capable of being enzymatically converted into a mass tag. The mass tag precursor may be part of a conjugate, a portion of which is a substrate for an enzyme. The mass tag precursor conjugate is typically converted by an enzme in a manner so that the resulting mass tag is localized in the vicinity of the enzyme. For example, the mass tag precursor may be water-soluble, wherein the enzyme catalyzes the formation of a water-insoluble mass tag which is deposited (e.g. precipitates) in the vicinity of the enzyme. The mass tag precursor may also include a substrate that is activatable by the enzyme to form a reactive species. Illustratively, the reactive species may be a short-lived reactive species that covalently binds to proximal to the enzyme. The mass tag precursor may also form a mass tag upon reaction with a reactive intermediate, for example a reative intermediate enzymatically formed.
Metal Heteroaryl Chelate: A complex comprising a metal having two or more separate binding interactions with one or more aryl groups containing at least one or more atoms other than carbon. Examples include mono-heteroaryl chelates in which none of A, A′, B, B′, C or C′ are bonded to another ring, biheteroaryl chelates, in which rings A and A′ are bonded together, with rings C and B being bonded together, and C′ and B′ bonded together, or terheteroaryl chelates, in which rings A, B, and C, are bonded together, and A′, B′, and C′ are bonded together.
Micelle: A micelle is a colloidal aggregate of large molecules, e.g., surfactant molecules.
Molecule of interest or Target: A molecule for which the presence, location and/or concentration is to be determined. Examples of molecules of interest include proteins, peptides, and nucleic acid sequences. In certain examples, the molecule of interest, or target, is a biomarker.
Monoclonal antibody: An antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of ordinary skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
MS: Mass spectrometry; mass spectrometric.
MSI: Mass spectrometric imaging.
Multiplex, -ed, -ing: Embodiments of the present invention allow multiple targets in a sample to be detected substantially simultaneously, or sequentially, as desired, using plural different conjugates. Multiplexing can include identifying and/or quantifying nucleic acids generally, DNA, RNA, peptides, proteins, both individually and in any and all combinations. Multiplexing also can include detecting two or more of a gene, a messenger and a protein in a cell in its anatomic context.
Nanoparticle: A nanoscale particle with a size that is measured in nanometers, for example, a nanoscopic particle that has at least one dimension of less than about 100 nm. Examples of nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle can produce a detectable signal, for example, through absorption and/or emission of photons (including radio frequency and visible photons) and plasmon resonance.
Neoplasia and Tumor: The process of abnormal and uncontrolled growth of cells. Neoplasia is one example of a proliferative disorder.
The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.” Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).
Photolabile Moiety: A moiety which is labile upon effective exposure to light. Photolabile moieties contain one or more bonds that are broken by exposure to light.
Photolysis: A chemical process in which a chemical compound is broken down by photons.
Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “residue” or “amino acid residue” refers to an amino acid that is incorporated into a protein, polypeptide, or peptide.
PR: Progesterone receptor.
Protein: A molecule, particularly a polypeptide, comprised of amino acids.
Proximal, -ly: The term “proximal” means being situated at or near the point of attachment or origin. Typically, attachment refers to coupling one moiety to another moiety. In some instances, “proximal” refers to the ability to distinguish single copies of target DNA with ISH. However, actual spatial resolution may be limited by the resolution achievable by the techniques used, such as spectrometric techniques. As used herein, proximal means within about 100 nm, within about 50 nm, within about 10 nm, or within about 5 nm. Proximal also may indicate within a range of about 10 angstroms to about 100 nm, about 10 angstroms to about 50 nm, about 10 angstroms to about 10 nm, or about 10 angstroms to about 5 nm.
Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, conjugate, or other active compound is one that is isolated in whole or in part from proteins or other contaminants. Generally, substantially purified peptides, proteins, conjugates, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, conjugate or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration. More typically, the peptide, protein, conjugate or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
Reactive Groups or functional groups: Formulas throughout this application refer to “reactive groups” or functional groups,” which can be any of a variety of groups suitable for coupling a first unit to a second unit as described herein. For example, the reactive group might be an amine-reactive group, such as an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acid chloride, such as sulfonyl chloride, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, and combinations thereof. Suitable thiol-reactive functional groups include haloacetyl and alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents, such as pyridyl disulfides, TNB-thiol, and disulfide reductants, and combinations thereof. Suitable carboxylate-reactive functional groups include diazoalkanes, diazoacetyl compounds, carbonyldiimidazole compounds, and carbodiimides. Suitable hydroxyl-reactive functional groups include epoxides and oxiranes, carbonyldiimidazole, N,N′-disuccinimidyl carbonates or N-hydroxysuccinimidyl chloroformates, periodate oxidizing compounds, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone-reactive functional groups include hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and combinations thereof. Active hydrogen-reactive compounds include diazonium derivatives, mannich condensation products, iodination reaction products, and combinations thereof. Photoreactive chemical functional groups include aryl azides, halogenated aryl azides, benzophonones, diazo compounds, diazirine derivatives, and combinations thereof.
Resonance: The appearance of delocalized electrons within certain molecules or polyatomic ions, so that the bonding cannot be expressed by one single Lewis formula. Typically used in reference to electron delocalization of molecules.
Sample: A biological specimen, typically containing DNA, RNA (including mRNA), protein, peptides, carbohydrates, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In one example, a sample includes a biopsy of an adenocarcinoma, a sample of noncancerous tissue, a sample of normal tissue (from a subject not afflicted with a known disease or disorder).
SILMSI: Stable isotope label-based mass spectrometry imaging.
Specific binding moiety: A member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 103 M−1 greater, 104 M−1 greater or 105M−1 greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Particular examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, glycoproteins, and protein A), nucleic acid sequences (for example, nucleic acid oligomers), and protein-nucleic acids. Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.
A substituent is an atom or group of atoms that replaces another atom in a molecule as the result of a reaction. The term “substituent” typically refers to an atom or group of atoms that replaces a hydrogen atom on a parent hydrocarbon chain or ring.
Substituted: A fundamental compound, such as an aryl or aliphatic compound, or a radical thereof, having coupled thereto, typically in place of a hydrogen atom, a second substituent. For example, substituted aryl compounds or substituents may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene. Again solely by way of example and without limitation, a long-chain hydrocarbon may have a substituent bonded thereto, such as one or more halogens, an aryl group, a cyclic group, a heteroaryl group or a heterocyclic group. Unless otherwise stated, any compound or functional group disclosed herein may be substituted or unsubstituted.
Tertiary Carbon: An sp3-hybridized (bound to four other atoms) carbon atom which is bound to three other carbon atoms and a fourth atom that is not carbon.
TOF: Time-of-flight; refers to time-of-flight mass spectroscopy in which ions' mass-to-charge ratio is determined via a time measurement.
Tyramine: A compound having the formula C8H11NO, also known as 4-(2-aminoethyl)phenol.
II. INTRODUCTIONDisclosed embodiments herein concern a method for imaging and/or quantifying one or more targets in a sample, e.g., a tissue sample such as a formalin-fixed, paraffin-embedded tissue sample, using mass tags and mass spectrometry (MS), such as laser desorption/ionization (LDI), including matrix-assisted laser desorption/ionization (MALDI). Some embodiments use an enzyme substrate and a separate mass tag precursor. The enzyme converts the substrate into an active species that can react with the mass tag precursor to deposit a mass tag, such as by precipitation, at the target site. Other embodiments use conjugates comprising a mass tag coupled directly or indirectly to an enzyme substrate moiety. The mass tag may be bonded to the enzyme substrate moiety by one or more covalent bonds, one or more ionic bonds, any other bonding association sufficiently robust to produce a conjugate suitable for use in the method, and combinations of bonds. The mass tag and the enzyme substrate moiety can be coupled directly together, or can be coupled using a linker.
In illustrative embodiments, a sample having a target of interest is first contacted with an enzyme, which may be coupled to a specific binding moiety that recognizes a particular target, or region of a target, such as an epitope. The sample is then exposed to the conjugate, or multiple conjugates. A mass tag is deposited at the site of a target in a sample at the target for subsequent detection by MS in the form of a mass code (an ion). The mass tags may be deposited (covalently, non-covalently, precipitated) at a target location, or multiple target locations, where they are exposed to ionization source to produce mass codes. The mass codes may be detected using mass spectrometry. For example, laser desorption ionization, matrix assisted laser desorption ionization, flowing atmospheric pressure afterglow ionization, laser ablation flowing atmospheric pressure afterglow ionization, desorption electrospray ionization, secondary ion, and laser ablation inductively coupled plasma mass spectrometry are technologies known in the art that could be adapted for use in tissue target identification using the mass tag conjugates. As such, disclosed embodiments herein are not limited to the method of mass spectrometry utilized for detection purposes.
The use of mass spectrometry and mass spectral imaging coupled to an enzymatic reaction enables identification, quantification, and imaging previously unavailable. In particular, the sensitivity, selectivity, quantifiability, and multiplexability of mass spectrometry is enhanced within the scope of the present disclosure by coupling with an amplification step. For example, the amplification step may include the use of an enzyme to create a large number of mass tags for a single recognition event. Furthermore, the selection of disclosed mass tags enhances the mass spectral analysis to enhance imaging and quantification. The current approach enables essentially limitless multiplexing. The use of isotopically labeled mass tags, which would be enzymatically amplified according to essentially the same kinetics, enables multiplexing that is also capable of providing relative quantitative information. This information is not only now uniquely available, but linked to contextual information through imaging which has heretofore be unavailable.
With respect to multiplexing, samples having multiple targets can be treated in a manner so that each target is identified and detected using a different mass. The mass tags may have the same chemical structure but include different isotopes and/or different amounts of a particular isotope such that mass tags are differtiable according to their sprectra. As described herein, because the kinetic relationship between various isotopes of a mass tag and the enzyme remains consistent, the amplification of the various isotopes will be consistent between targets. Accordingly, the methods described herein which use mass tags with the same chemical structure facilitate identification and at least relative target quantization.
III. MASS TAG PRECURSORSWhile mass spectrometry can be applied to a wide range of chemical species, the present disclosure provides examples of compounds with particular utility as mass tag precursors. While several examples of useful mass tags precursors are disclosed herein, one of ordinary skill will appreciate that these are merely examples selected to provide insight so that others may appreciate the entire genus of compounds useful within the scope of the present application. In particular, those compounds with closely related structures, properties, and functions are within the scope of the present disclosure.
One group of mass tag precursors useful within the scope of the present disclosure includes triarylmethane derivatives. Illustrative triarylmethane embodiments satisfy general Formulas 1A and 1B below.
With reference to general Formulas 1A, each R2, R4, and R3 may also comprise a linker, such as to link to an enzyme substrate moiety (e.g. a phosphate group, a lactam or a galactoside) to the triarylmethane. Each of the aryl groups can have various different substituents. For example, each of the R4 substituents can be the same or different. And each of R2-R4 independently can be the same or different.
Many of the triarylmethane derivatives include substituents at the para position relative to the tertiary carbon bearing R9. Substituents at thepara position help stabilize carbocation formation, either inductively through the introduction of alkyl groups, or by resonance through the introduction of electron-donating groups.
Certain amine-substituted compounds satisfy general Formulas 2A and 2B, below.
With reference to Formulas 2A and 2B, R1 and R1′ independently can be hydrogen, deuterium, alkyl, particularly lower alkyl, such as methyl, ethyl, propyl, butyl, etc., and aryl. The amine substituent can be located at any one or more of the ring positions. Thus, there can be multiple ring functionalizations with anywhere from zero to five substituents placed on each ring. Particular compounds include methyl at R1 and/or R1′, but a person of ordinary skill in the art will recognize that R1 and R1′ may be the same or different. In particular disclosed embodiments, R3 can be selected from hydrogen and alkylene oxy. In certain disclosed embodiments, R3 is at the para position and comprises an ethylene oxy subunit, or polyethylene glycol.
Scheme 1 illustrates a particular embodiment of the disclosed conjugate wherein triarylmethane mass tags 2 or 6 are exposed to a radiation source (e.g. a laser) to produce mass codes 4 or 8. A person of ordinary skill in the art will recognize that any compounds having either one of Formula 2A and 2B may undergo the process shown in Scheme 1.
In one embodiment, the mass tag precursor may have the following structure.
Another group of mass tag precursors classifiable as triarylmethane derivitives includes triarylmethane dyes. An exemplary triarylmethane dye satisfies general Formula 3.
With reference to Formula 3, R2, R3 and R4 also can be atoms, typically carbon atoms, in a ring bonded or fused to the illustrated rings. Each of the aryl groups can have various different substituents. For example, each of the R1 substituents can be the same or different. And each of R1, R1′, R2, R3, and R4 independently can be the same or different.
Compounds according to Formula 3 can be used in combination with a reducing agent, such as 5-bromo-4-chloro-3-indole generated from an AP catalyzed cleavage of 5-bromo-4-chloro-3-indole phosphate (BCIP), as illustrated in Scheme 2, to produce a mass tag for use according to disclosed embodiments. According to the embodiment of Scheme 2, an enzyme substrate 10, such as 5-bromo-4-chloro-indolyl-phosphate (BCIP), is contacted with an enzyme 12, such as alkaline phosphatase. This results in active species 14. In this particular example, species 14 is a reducing agent. Species 14 is contemplated to reduce a number of compounds, such as triarylmethane dye 18, during which, species 14 dimerizes to produce dimer 16. The reduction affords a colorless mass tag precipitate 20. Scheme 2 depicts using brilliant green. Precipitate 20 is converted into a mass code 22, such as by using a laser. A person of ordinary skill in the art will appreciate that dyes in addition to the brilliant green dye also can be used. When BCIP is used, a dimerized blue precipitate forms during the reaction of -5-bromo-4-chloro-3-indole with a triarylmethane compound having a structure as described in Formula 3. Laser-initiated ionization forms the corresponding mass code cation.
Such compounds typically satisfy the following Formula 4 as well.
With reference to this Formula 4, R1-R4 typically are hydrogen, lower alkyl, particularly methyl, ethyl, butyl, etc., aryl, aryl aliphatic, more typically aryl alkyl, more typically hydrogen, deuterium, methyl, ethyl, or phenyl. Exemplary compounds having such substitution patterns include, but are not limited to, the following compounds.
For certain disclosed embodiments, with reference to Formulas 3 and/or 4, R2-R4 can be substituents, typically comprising carbon atoms, but potentially heteroatoms too, such as nitrogen and/or oxygen, in a ring system. Particular disclosed embodiments may also have the following structures.
Another triarylmethane derivative useful within the present disclosure includes mass tag precursor conjugates including a naphthol moiety and a triarylmethane-diazo derivatives. Such compounds typically have the following general Formulas 5 and 6.
The hydroxyl and diazo moieties can be attached to the naphthalene ring at any position. The diazo moiety can be located at any position of the triarylmethane.
For certain disclosed embodiments, substituents on the triarylmethane rings are located at the para position relative to the tertiary carbon atom. Without being limited to a particular theory of operation, it is currently believed that having the substituents in this position may facilitate stabilizing the carbocation formed during analysis with mass spectrometry, such as by resonance or inductive effects. The hydroxyl group and diazo moieties can be attached to the naphthalene ring at any reasonable position, and any combination thereof. Particular embodiments have the general Formulas 7 or 8.
Naphthol-triarylmethane diazo compounds can be used to image a target in a sample. One disclosed embodiment of a method for using naphthol-triarylmethane diazo compounds is shown below in Scheme 3. According to this disclosed embodiment, a naphthol derivative 24 having a substrate moiety, such as a phosphate group, a galactoside, a lactam, etc., is reacted with an enzyme localized at a desired epitope using a specific binding moiety, such as an antibody. This produces a free naphthol 26, which can react with a water-soluble diazonium salt 34 that is produced by, for example, reacting an amine-triarylmethane compound 32 with an oxidizing agent, such as NaNO2. The triarylmethane-naphthol conjugate 28 is not water soluble, and precipitates at the target site. Specific embodiments of this type of conjugate include conjugates 36 and 38. Laser-initiated ionization produces detectable mass codes 30.
Naphthol-azo mass tag moieties can also be used in conjunction with a triaryl methane derivative to produce another class of photolabile mass tags. These mass tags can produce a mass code upon ionization of the mass tag precursor conjugate. The mass code produced from these mass tags is a naphthol-azo triaryl methane species having a tertiary carbocation. This mass code can be detected, such as by using mass spectrometry. In some embodiments, a naphthol azo moiety is bonded to an aryl ring of a triaryl methane derivative, as illustrated below.
A particular embodiment of this class of mass tags has the following structure.
Triarylmethane groups also can be used to form thio-based mass tag precursors. Particular disclosed embodiments have the following general Formula 9.
With reference to Formula 9, R10 can be aliphatic, such as alkyl, alkenyl, alkynyl, aryl, aryl aliphatic, more typically aryl alkyl, or any peptide containing a cysteine, such as glutathione.
In one embodiment, the mass tag precursor has the following structure:
Particular disclosed embodiments of the disclosed conjugate comprising thiophosphate-derived triarylmethane mass tag precursors can be made in any way known to a person of ordinary skill in the art, for example, the synthetic methods illustrated in Scheme 2.
With reference to Scheme 4, thiophosphates 40 and 48 are suitable substrates for reaction with alkaline phosphatase. Thioethers 56 and 62 are suitable for reaction with other enzymes, such as glutathione reductase and beta-lactamase, respectively. Reaction with the enzyme produces a thiol (RSH). The thiol group is a good nucleophile that can react with a triarylmethane compound 44 or 52, to form a triarylmethane-thiol mass tag 46, 54 or 60.
Thus, for example, a specific binding moiety may be coupled to an enzyme, such as alkaline phosphastase, beta-lactamase or glutathione reductase. Treating a sample with the enzyme-specific binding moiety conjugate then localizes the enzyme at a particular epitope in a sample. The sample is then contacted with a composition comprising a solvent-soluble triarylmethane compound, such as a water-soluble triarylmethane compound, which reacts with the enzymatically generated thiol to produce an insoluble mass tag precursor, which precipitates at the site of the target. Laser-initiated ionization then produces detectable mass codes, allowing imaging of the target in the sample.
Other disclosed embodiments concern using compounds, referred to herein as aryl-azo dyes, such as phenol-, naphthol-, or imidazole azo dyes, as indicated below in general Formulas 9-15. Y can include heteroatoms, such as nitrogen, oxygen, and sulfur; Z can include heteroatoms, such as oxygen and sulfur; and W can be any enzyme-reactive group prior to reaction with an enzyme (e.g. phosphate, lactam, or galactoside), or a group generated from an enzymatic reaction. With reference to general Formulas 10-16, W can be, for example and without limitation, hydroxyl or sulfhydryl, which is generated as a product of an enzymatic reaction, and can be located at any available ring position; R21 may be selected from aliphatic, aryl, heteroaryl, tyramine, and/or a tyramine moiety.
Certain disclosed embodiments also have the following general Formulas 17-19.
Certain disclosed embodiments use naphthol derivatives having the following general Formula 20 wherein the enzyme substrate moiety typically is at either C1 or C2.
Particular disclosed embodiments concern naphthol-azo mass codes produced from naphthol mass tag precursors having the following structures.
These particular disclosed embodiments of a mass tag precursosr are dephosphorylated with an enzyme, such as alkaline phosphatase, and then reacted with a diazo-containing compound (e.g. methoxy naphthol AS-TR) to produce the desired mass tag. The mass tag is ionized upon exposure to an energy source to produce the corresponding mass code.
Particular embodiments of naphthol-azo mass tags have the following structures.
As further illustrated in Scheme 5, certain embodiments have R5 being hydrogen, deuterium, or an electron donating group, R4 being hydrogen, deuterium, or an electron-withdrawing group, and R6 being hydrogen, deuterium, or an electron donating group. Scheme 5 illustrates using compound 66 comprising a naphthol portion and an azo portion derived from, for example, fast red. The naphthol-azo dye mass code 68 is produced via laser-initiated ionization.
Scheme 6 illustrates one embodiment of a naphthol-azo dye mass tag method.
A naphthol derivative 70, such as naphthol AS-TR, for use with alkaline phosphatase or naphthol-glucose for use with Beta-galactosidase, is provided. These derivatives are used in combination with the appropriate enzyme to cleave the enzyme substrate moiety and provide a naphthol derivative for reaction with a diazonium salt species. Scheme 6 illustrates the process using Fast Red KL as the diazonium salt. Diazonium salt 74 reacts with naphthol 72 to produce azo compound 76 suitable for laser-initiated ionization to form the detectable tag, 78. A person of ordinary skill in the art will appreciate that the structure of the naphthol derivative can vary, as can the diazonium salt structure.
Yet other embodiments utilize a photolabile nitrophenyl moiety, comprising at least one photolabile bond, as a mass tag precursor. These embodiments utilize a nitrophenyl moiety in the disclosed conjugate, particularly in a conjugate comprising tyramine or a tyramine derivative, and an optional linker or carrier, comprising a nitrophenyl linker and a mass code. A general formula illustrating these types of mass tag precursors is shown below.
With reference to Formula 21, R22-R24 independently are selected from aliphatic, heteroaliphatic, aryl, heteroaryl, carbonyl, halogen, hydrogen, hydroxyl, isothiocyanate, isocyanate, nitrile, nitro, thiol, or any combination thereof, with at least one of R22-R24 being nitro. More typically, R23 is nitro and is located at a carbon atom on the aromatic ring adjacent to the carbon atom bearing an R7 substituent. R7 is selected from aliphatic, or heteroaliphatic, more typically aliphatic, particularly —[CH(Me)]—. R8 is a linker, a charged species, a chromophore, or any combination thereof. More typically, R8 is a charged amino acid moiety, such as arginine, lysine, or histidine, or a tetra-alkyl ammonium species, such as tetramethyl ammonium, which is bound directly or via a linker to a chromophore, typically a dye or a hapten. In some embodiments, the R8 substituent has a m/z value in a range from about 100 to about 5000, more typically from about 500 to about 2000.
The mass code produced by nitrophenyl mass tags after exposure to an energy source typically has a formula [H2NR8]+. This mass code is produced either by photo-induced cleavage of a covalent bond between —[N(H)—R8] and the R7 substituent, or cleavage and subsequent ionization. The mass code is released from the mass tag and subsequently detected, such as by a mass spectrometer. Scheme 7 illustrates a particular disclosed embodiment wherein mas tag 80 is converted to mass code 82. Using a charged species as an R8 moiety, such as arginine, provides a cationic molecule at neutral pH. The cationic molecule is an MS detectable ion even in the absence of any matrix, thus illustrating the capacity of this mass tag to be used in conjunction with LDI, not specifically MALDI. In other embodiments, where R8 is not a charged moiety, the ionized mass code is produced by ionization, which takes place after bond cleavage.
Certain embodiments have a Formula 22, illustrated below. With reference to Formula 22, R22-R24, and R7 and R8 are as recited above.
Particular embodiments utilize nitrophenyl mass tags having the following structures.
In further illustrative embodiments concern photolabile a mass tag precursor conjugate comprising a heteroaryl moiety as the mass tag precursor. The photolabile characteristics of these moieties is described by Smith (Angew. Chem. Int. Ed. (2010)49:3612). These mass tag precursors have a general Formula 23, illustrated below.
With reference to Formula 23, R25-R28 independently are carbon or nitrogen, with at least one of R25-R28 being nitrogen; more typically R25-R28 are all nitrogen, thus comprising a tetrazine. A person of ordinary skill in the art will recognize that it is also possible for the heteroaryl moiety to comprise an azine, a diazine, or a triazine. R8 is a linker, a charged species, a chromophore, or any combination thereof. R8 can be selected from any of the recitations for R8 presented in reference to nitrophenyl mass tags. In particular embodiments, Y is sulfur. Scheme 8 illustrates a particular disclosed embodiment wherein mass tag 84 is exposed to a radiation source (e.g. a laser) and produces mass code 86.
Certain embodiments have a Formula 24, illustrated below. With reference to Formula 24, R8 can be selected from any of the recitations for R8 presented in reference to nitrophenyl mass tag precursors.
Particular embodiments have the following structure.
Certain disclosed embodiments of the substituted aryl and heteroaryl compounds satisfy general Formulas 25 and 26 below.
With reference to general Formulas 25 and 26, A in Formula 24 is selected from carbon and heteroatoms, such as oxygen and nitrogen. R12 is an amine (NH2, or NR1R1′) ether, thiol (SH), thioether, hydroxyl (OH), or combinations thereof. R13 is independently selected from aliphatic, such as alkyl, alkenyl, and alkynyl, aryl, aryl aliphatic, more typically aryl alkyl, more typically alkyl, and even more typically lower alkyl, such as methyl and ethyl, aryl, hydrogen, and combinations thereof. R13 also can be atoms in a ring connected with the first ring. With reference to Formula 26, the E ring is an aromatic or heteroaromatic ring having any suitable number of atoms in the ring, most typically 5, 6 or 7 atoms in the ring, and Y is a carbon or a heteroatom, such as nitrogen, oxygen or sulfur. For disclosed embodiments having a single ring, the amine substituents typically are either ortho or para to each other. Examples of dyes satisfying general Formula 25 for the heteroaryl compounds include, ortho and para phenylenediamine and diaminobenzidine (DAB).
Examples of disclosed embodiments having a second aromatic ring attached to the first include, without limitation, diaminonaphthalenes (DAN), such as those shown below.
A person of ordinary skill in the art will appreciate that heteroatoms, such as nitrogen, oxygen and sulfur, can be substituted for one or more carbon atoms in the aryl rings. Examples of such compounds include the aminoquinolines, as shown below.
Moreover, the aryl rings and the nitrogen atoms can include substituents other than hydrogen, including aliphatic and aryl groups, with lower alkyl groups being examples of suitable substituents.
Certain disclosed embodiments concern using oligomers generated by a peroxidase-catalyzed oxidative reaction detectable by MS. For example, DAB-stained tissue showed detectable signal (DAB dimer) but the background was high in some cases. 1,8-DAN gave a peak at m/z=297 and 8-AQ gave peaks at m/z=426 and m/z=567.
Tissue has also been stained with 1,8-DAN and 8-AQ. 8-AQ gave similar staining as DAB (Ki-67 on tonsil). A tissue sample stained by 8-AQ demonstrated that the oligomers (mostly trimers and tetramers) of 8-AQ can be readily detected on tissue samples without the need for using a matrix. Therefore, certain disclosed embodiments concern using oligomers of quinoline derivatives formed in an HRP-catalyzed reaction. These oligomers are highly colored and therefore absorb laser energy efficiently. In this sense, they serve as an in-situ matrix for their own ionization and desorption. In addition, the presence of multiple amines and nitrogen-containing aromatics facilitates protonation or the formation of radical cations, and therefore these oligomers can be readily ionized for use in MS.
The above-mentioned peroxidase substrates can also be used in conjunction with a metal ion. Examples of ions include Ni2+, Pd2+, Zn2+, Cu2+, Fe2+, Mn2+, Co2+, and others. A soluble metal salt solution, for example NiCl2 was pre-mixed with a peroxidase substrate solution, such as DAB or 1,8 DAN, and was applied to a tissue sample labeled with peroxidase on the target. Enhanced ionization of the mass tags are usually observed.
One advantage HRP offers is the irreversible inhibition of the enzyme by high concentrations of H2O2. Irreversible inhibition provides multiplexing capability by sequential deposition and inhibition steps.
In illustrative embodiments, photolabile, metal-complexed mass tags are sources of detectable mass codes, which can be used to determine the location of a particular target in a sample. Upon ionization of the mass tag precursor conjugate, a detectable mass code is produced. A person of ordinary skill in the art will recognize that ionization can involve cleavage of a covalent bond to produce a charged moiety or the release of a metal from a coordination complex to produce a charged moiety. In certain disclosed embodiments, a mass tag produces a detectable mass code upon ionization of a metal-heteroaryl chelate. In particular disclosed embodiments, metal-heteroaryl chelate mass tags produce mass codes in which a metal-biheteroaryl chelate is released from a mass tag precursor conjugate. Exemplary embodiments include formation of mass codes in which a metal is chelated to a bipyridine. Other embodiments include metal-heteroaryl chelate mass tags, which can produce mass codes in which a metal-terheteroaryl chelate is released from the mass tag precursor conjugate. These metal complexes can also contain one or more non-aryl ligands, such as, but not limited to nitrile, thiocyanate, hydroxyl, thiol, amine, carbonyl, phosphonyl, halide, phosphine, or heteroaliphatic. In particular examples, these chelates include mass codes in which a metal is chelated to a terpyridine. Scheme 10 illustrates a disclosed embodiment wherein mass tags 88 (bipyridyl) and 100 (terpyridyl) are ionized by a radiation source (e.g. a laser) to produce mass codes 102 and 104, respectively (see
A person of ordinary skill in the art will recognize that mass codes also can comprise metal-monopyridine chelates. Certain embodiments have mass tags with general Formulas 27-31, illustrated below.
With reference to Formula 27, L is selected from nitrile, thiocyanate, hydroxyl, thiol, amine, carbonyl, phosphonyl, halide, phosphine, or heteroaliphatic. Y and Z independently are selected from oxygen, sulfur, and —(nitrogen—R11)—. With reference to Formula 28, R29-R33 independently are selected from aliphatic, heteroaliphatic, aryl, heteroaryl, carbonyl, halogen, hydrogen, hydroxyl, isothiocyanate, isocyanate, nitrile, nitro, thiol, and any combination thereof. With reference to Formulas 27-31, M is a metal selected from Groups 3-12 of the periodic table, which is capable of forming a chelate structure; in some embodiments, M is Zn, Ni, Co, Ru, Cd, Pt, and Fe. M is selected to coordinate to rings A, A′, B, B′, C, C′. These rings can form a monoheteroaryl chelate, in which rings A, A′, B, B′, C, C′ are only chelated to M and not bonded to each other, a biheteroaryl chelate, in which rings A and A′ are bonded together, B and C are bonded together, and B′ and C′ are bonded together, or a terheteroaryl chelate, in which rings A, B, and C are bonded together and rings A′, B′, and C′ are bonded together. Y and Z independently are selected from oxygen, sulfur, and —(nitrogen—R11)—.
Other embodiments utilize peptide dimers as mass tags. These mass tags are produced from ionization of mass tag precursor conjugate comprising peptide dimers chelated to metal complexes. In certain embodiments, a tyramine or tyramine derivative is bound to a chelating reagent, such as nitrilotriacetic acid (NTA), which coordinates to a metal. The metal also coordinates to a peptide dimer mass tag comprising an amino acid capable of coordinating to a metal, such as histidine. Upon ionization, the peptide dimer is released and detected, such as by using mass spectrometry. Peptide dimer mass tags have a general formula (Formula 32) wherein R14 is selected from ether, hydroxyl, and —(nitrogen—R11)—, Y is selected from oxygen, sulfur, and —(nitrogen—R11)—, and R8 is as recited for nitrophenyl mass tag precursors and heteroaryl mass tag precursors.
Particular embodiments have the following structure.
Another class of mass tags that can be employed in the current method includes nanoparticles that are capable of forming a conjugate with an enzyme substrate, such as tyramine or tyramine derivatives. The nanoparticle typically comprises a semiconductor, a metal, or multiple metals selected from Groups 3-15, such as, but not limited to, Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, and Bi, or a combination thereof. Tyramine or tyramine derivatives can effectively couple to the metal of the nanoparticle. Upon ionization of the mass tag precursor conjugate, the metal cluster ions serve as the mass code, which can be detected using mass spectrometry.
In other embodiments, a nanoparticle is used in conjunction with a mass tag moiety and a tyramine or tyramine derivative moiety. In these particular embodiments, the nanoparticle serves as a carrier, which carries a mass tag and tyramine or tyramine derivative, or multiple such moieties, to the target. Certain embodiments utilize from 1 to about a few thousand ligands, more typically from 1 to about 1000 ligands, even more typically greater than 1 to several hundred, such as greater than 1 to about 500, greater than 1 to about 250, greater than 1 to about 200, greater than 1 to about 150, greater than 1 to about 100, and greater than 1 to about 50. In some embodiments, 1 to about 20 tyramine or tyramine derivatives are bound to the nanoparticle. Particular embodiments utilize ligands comprising an R15 moiety, a linker, a binding moiety, and any combination thereof. In certain embodiments, these ligands have a general Formula 33.
With reference to Formula 33, the R15 moiety is effectively coupled to a binding moiety. R15 is selected from any previously described mass tag moiety, a hapten, or a peptide, such as an oligo-peptide. R15 can be directly bonded to the binding moiety, or can be bound to the binding moiety with a linker. The linker can be aliphatic, heteroaliphatic, or heterobifunctional. The binding moiety is selected to effectively couple to the nanoparticle, and typically comprises a hydroxyl group, an amine group, a thiol group, or a phosphine group. Scheme 10 (
Particular embodiments use the following mass tag ligands.
Another class of mass tags useful for certain disclosed embodiments is metal ions that can be reduced to form metals, that precipitate at a particular target site. Typically, such metals are used in combination with a reducing agent to reduce a metal ion from a non-ground oxidation state (e.g. 1+, 2+, 3+, etc. oxidation state) to a ground state (oxidation state) for precipitation at a specified target. The metal can be irradiated with a laser to form ultra-small cluster ions (where ultra-small refers to the number of atoms in the cluster, which typically is from about 2 to about 10) and subsequently be detected by a suitable mass spectrometric technique. Solely by way of example, and without limiting the application to a particular theory of operation, the metals considered useful for this technique include bismuth, cobalt, copper, gold, indium, mercury, nickel, platinum, ruthenium, silver and tin. Further information regarding this subject matter can be found in U.S. Pat. No. 7,691,598, which is incorporated herein by reference. In addition, the description of enzymatic metal deposition disclosed in U.S. Pat. No. 6,670,113 and U.S. Patent Publication No. US-2008-0213783-A1 are incorporated herein by reference.
This embodiment can be illustrated with reference to using a horseradish peroxidase-silver ion reduction protocol in which silver nitrate is reduced to Ag0, which precipitates at the site of formation. In a particular embodiment, silver nitrate is reduced to Ag0 in the presence of hydroquinone (HQ), hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Other reducing agents include n-propyl gallate, 4-methylaminophenol sulfate, 1,4 phenylenediamine, o-phenylenediamine, chloroquinone, bromoquinone, 2-methoxyhydroquinone, hydrazine, and 1-phenyl-3-pyrazolidinone (phenidone aminophenol). Further information concerning this subject matter can be found in U.S. Pat. No. 7,183,072 , which is incorporated herein by reference. Additional enzymes that can be used include catalase and/or lactoperoxidase. Further information concerning enzyme species can be found in U.S. Pat. No. 7,592,153, which is incorporated herein by reference. The reduction of Ag0 by HQ is detailed in U.S. Pat. No. 7,632,652 (Column 9, line 20-50), which is incorporated herein by reference.
Ag2+ and Ag3+ ions are predominately detected from the deposited silver under LDI conditions. Silver deposited on a tissue sample can be imaged using MS.
Another example of a metal-based approach comprises using an alkaline phosphatase/5-bromo-4-chloro-3-indolylphosphate (AP-BCIP) reduction protocol. This method is illustrated below in Scheme 11. Similar imaging results can be achieved using this method. U.S. Pat. No. 7,087,379 teaches the use of a nitro blue tetrazolium (NBT)/BCIP method, which has been shown to result in an “increase in sensitivity for detection of nucleic acid sequences using alkaline phosphatase catalyzed detection techniques.” Page 5, lines 4-5 of U.S. Pat. No. 7,087,379.
Naphthols, 4-chloro-1-naphthol (4-CN) as an example, are known but not widely used substrates for HRP-based detection. For example, 1-naphthol has been used as the substrate for HRP, but without using MS techniques. See, for example, Histochemistry (1985) 83:97-102. The naphthol precipitate is further stained with various basic dyes (cationic), such as azure A, methylene blue, cresyl violet, crystal violet, methyl green, acridine orange, and basic fuchsin. Certain embodiments concern using the dyes absorbed to naphthol precipitates in mass tag applications.
Basic Dyes Adsorbing to 1-Naphthol PrecipitateThese dyes are mostly positively charged and have high absorbance at the wavelength of the laser. Therefore they are useful candidates for use as mass tags. Crystal violet has been used in AP-BCIP based systems. These dyes presumably physically adsorb to the naphthol precipitate instead of becoming chemically conjugated to the naphthol precipitate. However, they have been shown to be stable and specific. A tissue sample stained by such a method may be imaged using mass spectrometry.
This approach can be used either sequentially, or in a one-step method. The one-step method involves mixing naphthol and the dye first before adding to a tissue sample. In this way, the dye would precipitate together with naphthol as the enzymatic reaction proceeds, as shown below in Scheme 12 (
The adsorbed mass tag, for example crystal violet, can be readily detected by LDI MS, even on normal glass slides (non-ITO slide). In this approach, many non-triarylmethane molecules can be used as mass tags.
In futher illustrative embodiments, one or more atoms of any mass tag precursor, mass tag, and/or mass code disclosed herein, or hereafter discovered that is useful for practicing the disclosed method, may be replaced with stable isotopes (e.g., 2H, 3H, 13C, 14C, 15N, 18O etc.). Replacing atoms with stable isotopes can be used, for example, to provide a homologous series of mass tags having the same chemical structure but differing molecular weights. For example, one or more hydrogen atoms on one or more rings of a triarylmethane-containing compound, an aryl azo dye, a basic dye, or a heteroaryl compound as described herein can be replaced by deuterium or tritium atoms. Similarly one or more carbon atoms can be replaced by one or more carbon-13 atoms. In some embodiments, one or more isotopes may be present in an enzyme substrate, e.g., a naphthol (such as a naphthol phosphate), that reacts with an enzyme and a mass tag precursor to form a mass tag. Desirably, the isotopes are in the portion of the mass tag that forms the mass code after ionization, thereby providing mass codes having the same chemical structure but differing masses.
Additional species of triaryl methane derivatives are disclosed in U.S. Pat. No. 6,780,981 and types of mass spectrometry techniques are disclosed in U.S. Pat. No. 7,198,893, where are incorporated by reference herein. Compounds disclosed herein include reference to substituents by “R” groups or substituents, such as “R1”. Unless otherwise indicated, R1 and R1′ may be selected from aliphatic, such as alkyl, more typically lower alkyl, such as methyl, ethyl, propyl, butyl, etc., alkenyl, alkynyl, aryl, aryl aliphatic, such as aryl alkyl, hydrogen, and deuterium; R2, R3, R4, R5 and R6 may be selected from aliphatic, such as alkyl, more typically lower alkyl, such as methyl, ethyl, propyl, butyl, etc., alkenyl, alkynyl, aryl, aryl aliphatic, such as aryl alkyl, and electron-donating heteroatom-containing moieties selected from, but not limited to, ether (RaORb), hydroxyl (RaOH), silyl ether (RaRbRcSiORd), phosphine (PRaRbRc), thiol (RaSH), thioether/sulfide (RaSRb), disulfide (RaSSRb), isothiocyanate (RaNCS), isocyanate (RaNCO), amine (NH2, NHRa, NRaRb), amide (RaNRbC(O)Rc), ester (RaOC(O)Rb); R20 is an electron withdrawing group, and may be selected from heteroatom-containing moieties selected from halogen (I, Br, Cl, F), carbonate (RaOC(O)ORb), carboxyl (RaC(O)OH), carboxylate (RaCOO−), ester (RaC(O)ORb), ketone (RaC(O)Rb), phosphate (RaOP(O)OH2), phosphoryl (RaP(O)(OH)2), sulfinyl (RaS(O)Rb), sulfonyl (RaSO2Rb), carbonothioyl (RaC(S)Rb or RaC(S)H), sulfino (RaS(O)OH), sulfo (RaSO3H), amide (RaC(O)NRbRc), azide (N3), nitrile (RaCN), isonitrile (RaN+C−), and nitro (RaNO2). With reference to all the heteroatom-containing moieties disclosed herein, Ra represents one or more of the aryl rings of the triarylmethane, the naphthol, or the diazo mass tag precursors/mass tags/mass codes described herein; and Rb, Rc, and Rd independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and any combination thereof. Unless otherwise stated, R9 can be any group that can form, or that can be removed, to form, an ion, typically a carbocation. Exemplary R9 groups can include halogens (fluorine, chlorine, bromine and iodine), nitriles (CN), sulfonyl groups, such as mesyl (Ms), tosyl (Ts), trifluoromethyl (Tf), hydroxyl, ether, amine, thiol, thioether, and salts thereof. Unless otherwise stated, R19 is any group that can form, or that can be removed to form, a carbocation. Typically, R19 is selected from oxygen, sulfur, and —(nitrogen—R11)—.
Any one or more of R1, R1′, R2, R3, R4, R5, and R6, particularly R2, R3, R4, R5, and R6, may be varied to produce a compound of a particular selected molecular weight. For example, R1, R1′, R2, R3, R4, R5, and/or R6 may be a species, such as halogen or a stable isotope, such as deuterium, and may be used to provide a homologous series of tags of differing molecular weights. In some embodiments, one or more ring carbon atoms may be replaced with one or more 13C and/or 14C isotopes to provide a homologous series of tags of differing molecular weights. Unless otherwise indicated, R16 is selected from ether, hydroxyl, —(nitrogen—R11)—; R17 is selected from aliphatic, heteroaliphatic, aryl, and heteroaryl; Y is selected from oxygen, sulfur, and —(nitrogen—R11)—; and R11 is selected from aliphatic, heteroaliphatic, aryl, heteroaryl, and hydrogen.
IV. TYRAMINE AND TYRAMINE DERIVATIVESIn some embodiments, mass tag precursors are conjugated to a tyramine or a tyramine derivative. Particular embodiments utilize tyramine for the formation conjugates. A mass tag precursor conjugate comprising a tyramine or tyramine derivative typically have the following general Formula 34.
Some embodiments utilize tyramine derivatives for mass tag precursor conjugate. These tyramine derivatives have the following general Formula 35, wherein n is from 1 to 20.
Certain embodiments include the use of a linker, which is used to couple one moiety to a second moiety. For example, linkers may be used to couple, such as to covalently or electro-statically bind, a mass tag precursor to a tyramine or a tyramine derivative. These linkers can be selected from any moiety capable of effectively coupling two moieties, such as a mass tag precursor and a tyramine or tyramine derivative, including aliphatic, heteroaliphatic, or heterobifunctional linkers.
In particular embodiments, the optional linker can have the following Formula 36.
With reference to Formula 36, each X independently is selected from CH2, oxygen, sulfur, and —(nitrogen—R11)—. R18 is carbonyl or sulfoxyl; n is 1-20; and p is 0 or 1.
Other embodiments utilize polyethylene glycol linkers, having a general formula PEGn, where in n is 1-20, typically 4 to 12.
VI. CARRIERSCertain embodiments utilize carriers as a way to deliver multiple mass tags, tyramine moieties, or tyramine derivatives to the target. Carrier moieties can facilitate the deposition of the mass tag precursor proximal to the target. In particular disclosed embodiments, a carrier may be used to facilitate formation of covalent bonds of mass tag precursor conjugates comprising tyramine or a tyramine derivative to the target, providing a potential increased signal produced by the mass code upon cleavage and ionization.
Carriers can be selected from polymers, biomolecules, liposomes, micelles, or nanoparticles. Exemplary polymers include linear polymers, such as polyacid (more typically polyacrylic acid), polyamine (more typically poly-L-lysine), polysaccharide (more typically dextran, or chitosan), polyhydrazine, or a co-polymer. Other exemplary polymers are hyperbranched polymers, such as polyethyleneimine or a dendrimer. Exemplary biomolecules include proteins, polypeptides, oligopeptides, peptides, nucleic acids, DNA, RNA, oligosaccharides, polysaccharides, and monomers thereof. These carriers are selected to bind individual or multiple mass tag moieties, tyramine moieties, and/or tyramine derivative moieties. Typically binding occurs via thiol, amine, carboxylic acid, hydroxyl, halide, anhydride, or hydrazine moieties located on the polymer and/or biomolecule.
Nanoparticle carriers typically comprise a semiconductor material, a metal, or multiple metals. Metals are selected from Groups 3-15 of the periodic table, such as, but not limited to, Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, and Bi, or a combination thereof. Nanoparticles are typically bound to a mass tag ligand, tyramine and/or tyramine derivative, or multiple such moieties, through a binding moiety, such as a thiol group, an amine group, a hydroxyl group, or a phosphine group located on the mass tag, tyramine or tyramine derivative.
VII. EMBODIMENTS OF A MASS TAG PRECURSOR CONJUGATECertain embodiments concern a mass tag precursor conjugate comprising an enzyme substrate moiety, a mass tag precursor, and an optional linker. For example, an enzyme substrate moiety can be either conjugated to mass tag precursor disclosed herein or hereafter developed, or an enzyme substrate moiety may be conjugated to the mass tag precursor using a linker. Certain disclosed conjugates satisfy general Formula 37 below.
Enzyme Substrate MoietyOptional LinkMass Tag Precursor Formula 37
With reference to general Formula 37, the mass tag precursor can be any tag useful for imaging targets as disclosed herein, and that is readily ionizable and detectable under LDI conditions. The enzyme substrate moiety can be any group capable of being transformed by an enzyme. The optional linker can be any structure that does not interfere with the conjugate for use in disclosed embodiments of the process for imaging targets. In some embodiments, the linker is hydrophobic. In more particular embodiment, the linker is an alkylene oxy moiety, such as an ethylene oxy subunit, or polyethylene glycol group.
Certain disclosed embodiments concern a mass tag precursor conjugate comprising a mass tag precursor, tyramine or a tyramine derivative, and an optional linker or carrier. These conjugates have the following general Formula 38.
With reference to Formula 38, n is 1-20. The optional linker is selected from aliphatic, heteroaliphatic, or heterobifunctional. The mass tag is a moiety that produces a mass code upon ionization of the conjugate.
The previously disclosed mass tags and tyramine or tyramine derivatives can be used to form conjugates, which can be used in embodiments of the disclosed method.
In illustrative embodiments, the conjugate comprises a triarylmethane group mass tag precursor, and have a general Formula 39. The optional linker may be selected from aliphatic, aryl, heteroaliphatic, and combinations thereof. In particular disclosed embodiments, the linker is an alkylene oxy linker, which provides a pendent hydroxyl group for attaching an enzyme substrate moiety, such as a phosphate group. A person of ordinary skill in the art will appreciate that these particular linkers can include heteroatoms other than oxygen, such as sulfur. Sulfur is particularly suitable for coupling beta-lactams, such as cephalosporin, to a triarylmethane mass tag. Other linkers that can be used include any short, hydrophobic linker.
With reference to Formula 39, a person of ordinary skill in the art will appreciate that an enzyme substrate moiety can be attached to the desired linker or directly to the aryl ring. The triarylmethane can be selected to provide a first-water soluble tag coupled to an enzyme substrate moiety. For example, certain disclosed compounds include a phosphate group or a galactoside, as an enzyme substrate moiety coupled to an aryl ring, such as through a pendent hydroxyl group. The phosphate and/or galactoside is water soluble. In addition the triarylmethane derivatives can be coupled to a substrate suitable for reaction with a lactamase, such as a lactam. Removing the phosphate, galactoside, and/or lactam group, such as by an enzymatic reaction using an appropriate enzyme (e.g. a phosphatase cleaves phosphates, a galactosidase cleaves galactosides, and a lactomase cleaves lactams), forms an insoluble compound that precipitates at the site of the target. Laser-initiated ionization forms the mass code. Particular disclosed embodiments have Formulas 40-42
Certain disclosed embodiments concern using a mass tag precursor conjugate having the following Formulas 43-45, wherein n ranges from 1 to about 10.
Scheme 13 provides an example of a conjugate comprising a mass tag precursor and an enzyme substrate and a method for making the conjugate.
According to Scheme 13, a protected monosaccharide 120, is reacted with triarylmethane derivative 122, in the presence of 0.5 equivalent of indium bromide (InBr3), to form ether 124. Ether 124 is then deprotected in 20% hydrazine/methanol to form deprotected conjugate 126 comprising both a triarylmethane mass tag and a substrate for a galactosidase. The conjugate 126 is an exemplary conjugate that may be a substrate for β-D-galactosidase, which catalyzes the hydrolysis of galactosides into monosaccharides. If the galactoside is an α-galactoside, the enzyme is a-galactosidase, and if the galactoside is a β-galactoside, the enzyme is β-galactosidase. Thus, as shown in Scheme 13, contacting conjugate 126 with a enzyme (e.g. galactosidase) produces a monosaccharide, such as D-galactose 130, and a water insoluble mass tag 128 that can be ionized for detection by a suitable MS technique.
Scheme 14 also provides an example of a conjugate comprising a mass tag precursor and an enzyme substrate. Conjugate 132 comprises a mass tag precursor and a β-lactam moiety, such as cephalosporin, as a substrate for β-lactamase. β-lactams have a common element in their molecular structure: the four-atom β-lactam ring. Additional examples of β-lactams include penicillins, cephamycins, and carbapenems (ertapenem). The lactamase enzyme cleaves the conjugate to form the water insoluble mass tag 134, which upon ionization produces a mass code detected by a suitable MS technique.
Some disclosed embodiments use triarylmethane derivatives as mass tag precursors to construct the mass tag precursor conjugate. Certain embodiments of the conjugate have a general Formula 46, illustrated below.
With reference to Formula 46, optional linkers include aliphatic, heteroaliphatic, or heterobifunctional linkers; and n ranges from 1 to about 20. In some embodiments, mass tag precursor conjugates are formed with a carrier, which allows for one or more mass tags, tyramine and/or tyramine derivatives to be deposited at a target. Carriers are selected from polymers, biomolecules, and nanoparticles.
Particular embodiments of triarylmethane mass tags have the following Formula 47.
With reference to Formula 47, R7, n, and the optional linker are as recited above. R4 and R5 independently are selected from aliphatic, heteroaliphatic, aryl, heteroaryl, and hydrogen.
Particular embodiments concern using a mass tag precursor conjugate having the following structure.
Further illustrative embodiments concern a conjugate comprising an enzyme substrate moiety that is either covalently bound to an azo-naphthol mass tag precursor or bound through a linker. Certain disclosed embodiments satisfy general Formula 48 below.
With reference to general Formula 48, the enzyme substrate moiety can be any group capable of being cleaved by an enzyme. The optional linker can be any hydrophobic, ethylene oxy subunit, or polyethylene glycol group capable of binding the enzyme substrate moiety. The diazo species and the ether containing the enzyme substrate moiety can be located at positions 1, 2, and/or 4, or any combination thereof. Certain disclosed embodiments of naphthol azo dye conjugates have the Formulas 49-51 indicated below.
Particular disclosed embodiments have the following Formulas 52-54.
Another genus of the disclosed mass tag precursor conjugate includes those in which the mass tag precursor is a naphthol-azo moiety. These embodiments of the disclosed conjugate have a general Formula 55, illustrated below.
With reference to Formula 55, Z is selected from oxygen sulfur, and —(nitrogen—R11)—. In particular embodiments, Z is oxygen.
A particular embodiment of the mass tag precursor conjugate is illustrated below.
In some embodiments, naphthol-azo mass tag moieties are used in conjunction with a triaryl methane derivative to produce another class of photolabile conjugates. In these examples, a naphthol azo moiety is bonded to an aryl ring of a triaryl methane derivative, as illustrated below.
The mass tag precursor conjugate may deposit a mass tag, which forms a mass code comprising a naphthol-azo-triarylmethane cation. This cationic mass code is then detected, typically using mass spectrometry. A particular embodiment of these conjugates is illustrated below.
In illustrative embodiments, a conjugate comprises a photolabile nitrophenyl moiety as a mass tag precursor. These embodiments utilize a nitrophenyl mass tag moiety as a linker to effectively couple a mass code precursor to the mass tag precursor conjugate. A general formula illustrating these types of conjugates is shown below.
With reference to Formula 57, the optional linker typically is selected from aliphatic, heteroaliphatic, or heterobifunctional linkers and n ranges from 1 to about 20. R22-R24 independently are selected from aliphatic, heteroaliphatic aryl, heteroaryl, carbonyl, halogen, hydrogen, hydroxyl, isothiocyanate, isocyanate, nitrile, nitro, thiol, and any combination thereof, with at least one of R22-R24being nitro; R7 is aliphatic, or heteroaliphatic. R8 is selected from a linker (typically aliphatic or heteroaliphatic) a charged species (typically selected from charged amino acids, such as arginine, lysine, or histidine), a tetra-alkyl ammonium species (such as tetramethyl ammonium), a chromophore (typically selected from a dye or a hapten) and any combination thereof. In particular embodiments, R8 moieties have a m/z value in a range from about 100 to about 5000, or from about 500 to about 2000.
Certain embodiments have a Formula 58, illustrated below, where the variables are as recited for Fomula 57.
Particular embodiments of these conjugates are illustrated below.
Some photolabile mass tag precursor conjugates comprising a tyramine or tyramine derivative include heteroaryl moieties as mass tag precursors. These conjugates have a general Formula 59, illustrated below.
With reference to Formula 59, the optional linker typically is selected from aliphatic, heteroaliphatic, or heterobifunctional. R25-R28 independently are selected from carbon or nitrogen, with at least one of R25-R28 being nitrogen. In certain embodiments, R25-R28 are nitrogen. A person of ordinary skill in the art will recognize that when R25-R28 are nitrogen, the heteroaryl moiety is a tetrazine. A person of ordinary skill in the art also will appreciate that the heteroaryl moiety can be an azine (where one of R25-R28 comprises nitrogen), a diazine (where two of R25-R28 comprises nitrogen), or a triazine (where three of R25-R28 comprises nitrogen). R8 is selected from a linker (typically aliphatic or heteroaliphatic) a charged species (typically selected from charged amino acids, such as arginine, lysine, or histidine; or a tetra-alkyl ammonium species, such as tetramethyl ammonium) a chromophore (typically selected from a dye or a hapten) and any combination thereof. In particular embodiments, R8 moieties have a m/z value in a range from about 100 to about 5000, or from about 500 to about 2000.
The mass codes produced from these embodiments of the disclosed conjugate, after exposure to an energy source, typically have a formula NCS—R8, wherein R8 can be a charged moiety. In cases where R8 is not a charged moiety, the mass code is obtained by bond cleavage, which first produces a neutral NCS—R8 fragment, and subsequent ionization produces a charged mass code. This mass code results from photo-induced degradation of the heteroaryl moiety. The mass code subsequently is detected, such as by a mass spectrometer.
Particular embodiments have a structure illustrated below.
In some embodiments, photolabile the metal-complexed mass tag precursor conjugate is a source of a detectable mass code, which can be used to determine the location of a particular target in a sample. Exemplary embodiments utilize metal complexes as mass tags. Upon ionization of the conjugate, a detectable mass code is produced. A person of ordinary skill in the art will recognize that ionization can involve cleavage of a covalent bond to produce a charged moiety or the release of a metal from a coordination complex to produce a charged moiety.
Certain embodiments have a mass tag precursor conjugate with a general Formulas 60 and 61, illustrated below.
With reference to Formulas 60 and 61, the optional linker typically is selected from aliphatic, heteroaliphatic, or heterobifunctional linkers. With reference to Formula 60, L is a non-aryl ligand, such as, but not limited to nitrile, thionitrile, hydroxyl, thiol, amine, carbonyl, phosphonyl, halide, phosphine, or heteroaliphatic.
With reference to Formula 61, R29-R33 independently are selected from aliphatic, heteroaliphatic, aryl, heteroaryl, carbonyl, halogen, hydrogen, hydroxyl, isothiocyanate, isocyanate, nitrile, nitro, thiol, and any combination thereof. For both Formulas 60 and 61, M is a metal selected from Groups 3-12 of the periodic table capable of forming a chelate structure; in some embodiments, M is Zn, Ni, Co, Ru, Cd, Pt, and Fe. M is coordinated to rings A, A′, B, B′, C, C′. These rings can form a monoheteroaryl chelate, in which rings A, A′, B, B′, C, C′ are only chelated to M and not bonded to each other; a biheteroaryl chelate, in which rings A and A′ are bonded together, B and C are bonded together, and B′ and C′ are bonded together; or a terheteroaryl chelate, in which rings A, B, and C are bonded together and rings A′, B′, and C′ are bonded together. Z is selected from oxygen, sulfur, and —(nitrogen−R11)—.
Particular embodiments of this genus of mass tag precursor conjugate are illustrated below.
In other embodiments utilizing a metal-heteroaryl chelate mass tag precursor conjugate, a tyramine or tyramine derivative is bound to a chelating reagent, such as nitrilotriacetic acid (NTA), which coordinates to a metal. The metal also coordinates to a peptide dimer comprising an amino acid capable of coordinating to a metal, such as histidine. Upon ionization, the peptide dimer is released and detected, such as by using mass spectrometry. Particular embodiments have the following general formula.
With reference to Formula 62, M and Z are as recited above. The amino acid moieties can be an amino acid capable of forming a covalent or ionic bond with a metal (such as, but not limited to histidine), and R8 is selected from a linker (typically aliphatic or heteroaliphatic), a charged species (typically selected from charged amino acids, such as arginine, lysine, or histidine; or a tetra-alkyl ammonium species, such as tetramethyl ammonium), a chromophore (typically selected from a dye or a hapten) and any combination thereof. In particular embodiments, R8 moieties have a m/z value in a range from about 100 to about 5000, or from about 500 to about 2000.
In other illustrative embodiments, the mass tag precursor is a nanoparticle-based mass tag precursor conjugate. In particular disclosed embodiments, the mass tag precursor comprises an enzyme substrate, such as a tyramine or tyramine derivative. In these embodiments, the nanoparticle may be used as a carrier to carry a mass tag and tyramine or tyramine derivative, or multiples of such moieties to the target. Alternatively, the nanoparticle, itself, can be used as a mass tag. When a conjugate utilizes a nanoparticle as a carrier, the mass tag typically is a ligand, which produces a mass code upon ionization. Disclosed embodiments of these genus of the conjugate have a Formula 63, illustrated below.
With reference to Formula 63, a ligand typically comprises a R15 moiety, a linker, and a binding moiety. R15 can be selected from any of the previously disclosed mass tags, a hapten, or a peptide. Linkers are selected from aliphatic, heteroaliphatic, and heterobifunctional linkers; and n ranges from 1 to about 20. Binding moieties are groups that are selected to bind to the nanoparticle, and typically are selected from a hydroxyl moiety, an amine moiety, and a thiol moiety and q is 1 to about 2000 (more typically 1 to about 500). The nanoparticle is selected to contain a semiconductor, a metal or multiple metals. Metals are typically selected from Groups 3-15 of the periodic table, more typically, Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, and Bi, and a combination thereof. Tyramine is represented when R16 is hydroxyl, R17 is aliphatic, n is 2 and Y is NH.
In particular embodiments, a nanoparticle-based mass tag precursor conjugate has any one of Formulas 64 and 65. The variable “q” is 1 to about 2000 (more typically 1 to about 500); and r is 1 to about 20.
Nanoparticles also can be used as mass tags in disclosed embodiments. When a nanoparticle is used as a mass tag, the metal cluster ions generated upon irradiation act as a mass code, which is detected, such as by mass spectrometry. A nanoparticle mass tag typically is conjugated to a tyramine or a tyramine derivative to provide a conjugate such as that illustrated in Formula 66. With reference to Formula 66, n and r are as recited in Formulas 64 and 65.
In particular disclosed embodiments, the mass tag precursor conjugate reacts with an enzyme to produce a mass tag that is deposited proximal to the target. In particular embodiments, the enzyme reacts with the enzyme substrate to cleave a particular moiety from the mass tag precursor conjugate to form the mass tag. In other disclosed embodiments, the enzyme reacts with the mass tag precursor conjugate to form an active species, such as a radical, which allows the mass tag to deposit proximal to the target.
IX. SPECIFIC BINDING MOIETY/ENZYME CONJUGATESParticular embodiments also concern conjugates comprising an enzyme coupled, typically being covalently bound, to a specific binding moiety, as shown in general Formula 67. The enzyme can be any enzyme capable of recognizing an enzyme substrate moiety connected to a mass tag or other species. The specific binding moiety can be any species capable of binding to a target or an epitope.
Enzyme—Specific Binding Moiety Formula 67
The specific binding moiety can be a protein, a peptide, an antibody, an oligomer, a lectin, and/or a nucleic acid, such as DNA or RNA, as indicated below.
Enzyme—Protein Enzyme—DNA
Enzyme—Antibody Enzyme—RNA
The enzyme is bound to any specific binding moiety capable of specifically recognizing a target. Certain particular embodiments concern alkaline phosphatase, galactosidases, and lactamases conjugated to specific binding moieties, as shown below.
Alkaline Phosphatase—Specific Binding Moiety
Galactosidase—Specific Binding Moiety
β-lactamase—Specific Binding Moiety
Certain disclosed embodiments of specific binding moiety/enzyme conjugates include an antibody covalently bound to alkaline phosphatase, shown in Formula 68. This conjugate can be used to enzymatically cleave an enzyme substrate moiety, which will subsequently react with a mass tag.
Alkaline Phosphatase—Antibody Formula 68
Certain disclosed embodiments of mass tags, such as triarylmethane-base compounds are commercially available. Other embodiments can be made as will be recognized by a person of ordinary skill in the art. Scheme 15 illustrates one embodiment of a method for making particular triarylmethane compounds that are useful as mass tags according to certain disclosed embodiments. As illustrated in
Scheme 15, starting compound 136, 2-(4-bromophenoxy)ethanol, is commercially available, such as from Aldrich Chemical Co. Compound 136 is converted to the lithiated derivative 138 by reaction with n-butyl lithium. Compound 138 is a good nucleophile for reaction with carbonyl derivatives, e.g. compound 140, such as 4,4′-(dimethylamino)benzophenonone. A person of ordinary skill in the art will recognize that any commercially available benzophenone derivatives may be used, such as, but not limited to, 4,4′-bis(dimethylamino)benzophenone, 4,4′-bis(diethylamino)benzophenone, 4,4′-dimethoxybenzophenone, 4-benzoylbenzoic acid,bBenzophenone-4-isothiocyanate, and 4-aminobenzophenone. Reaction with compound 140 produces hydroxylated triarylmethane derivative 142, which can be converted into the corresponding nitrile compound 144 by reaction with potassium cyanide (KCN). Compound 144 is then converted to conjugate 146, which is suitable for an enzymatic reaction, using phosphorous oxychloride (POCl3).
Scheme 16 illustrates one embodiment for a method for making a substrate both for horseradish peroxidase (HRP) and for alkaline phosphatase. Compound 148, 2-methylbenzothiazole, commercially available from Aldrich, is reacted with para-anisaldehyde 150 (4-methoxybenzaldehyde, which also is commercially available from Aldrich), to form olefin 152. Olefin 152 is then reacted with hydroiodic acid (HI) to form phenol 154. Phenol 154 is a suitable substrate for HRP. Phenol 154 also can be converted into a substrate suitable for alkaline phosphatase by reacting phenol 154 with phosphorous oxychloride (POCl3) to produce phosphate 156.
Scheme 17 illustrates one embodiment of a method for making a substrate for -lactamase. In this example, a cephalosporin derivative 158 is reacted with compound 160 to produce a substrate suitable for a -lactamase 162.
Scheme 18 illustrates one embodiment for synthesizing nitrophenyl mass tag precursors. As shown in Scheme 18, nitrophenyl mass tag compounds can be synthesized using solid phase synthesis. A nitrophenyl compound 166 is loaded onto a trityl chloride resin 164. The Fmoc protecting group of protected amine 168 is removed under basic conditions to provide an amine 170, which is further coupled using peptide coupling conditions to give rise to an amino acid-coupled amine 172. This step can be repeated to conjugate as many amino acids or linkers as desired, in particular to give rise to peptide 174. The desired mass tag precursor 176 is liberated from the resin under acidic conditions.
Scheme 19 illustrates one embodiment of a method for making a particular mass tag precursor conjugate. In this scheme, a nitrophenyl mass tag is coupled to a tyramine through a coupling sequence. Carboxylic acid 176 is subjected to an esterification reaction using dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide to provide activated ester 178. Ester 178 can then be converted to the mass tag precursor conjugate 180 using tyramine.
Scheme 20 illustrates one embodiment of a method for making a particular mass tag precursor conjugate. In Scheme 20, a commercially available carboxylic acid 182 is coupled to an esterified tyramine moiety 184. The resulting amide 186 is chelated with nickel to provide chelate 188, followed by chelation of the nickel to a histidine dimer 190 to give mass tag 192.
One embodiment of a method for making a stable-isotope-labeled mass tag precursor pair capable of reacting with naphthol phosphate in the presence of a phosphatase, e.g., alkaline phosphatase, to produce aryl azo dyes is illustrated below in Scheme 21. The mass tag precursor pair is suitable for use in a multiplexed assay, such as a duplexed assay, to detect and quantify two biomarkers, or two forms of a single biomarker, in a tissue sample. An isotopically-labeled mass tag precursor pair can be used, for example, to detect relative amounts of ER and PR breast cancer biomarkers in a tissue sample. In some embodiments, the isotopically-labeled mass tag precursor pair can be used to detect two forms of a biomarker, such as intact Her2 and truncated Her2.
Aniline 194 and deuterated aniline analogs, such as aniline-d5 198, are converted to benzene diazonium cations 196 and 200, respectively, by reaction with sodium nitrite and acid (e.g., tetrafluoroboric acid) to produce a “light” diazonium salt that may be used to produce a “light” mass tag precursor and a “heavy” diazonium salt that may be used to produce a “heavy” mass tag precursor:
The diazonium salts react with a naphthol phosphate in the presence of a phosphatase enzyme to produce an azo dye mass tag precursor, which precipitates as a mass tag, which is ionizable to produce reporter ions, or mass codes, with the same chemical structure but having molecular weights that differ by as little as 1 Dalton, and perhaps 2-5 daltons (Scheme 22).
Other deuterated aniline analogs also may be used. Suitable deuterated aniline analogs have the general formula
where n is 1, 2, 3, 4, or 5; preferably n is 2-5.
In another embodiment, Fast Blue BB can be used to prepare stable-isotope-labeled diazonium analogs capable of reacting with a naphthol phosphate, such as in a multiplexed assay to detect and quantify multiple biomarkers. Fast Blue BB analogs have a general formula
where n is 0, 1, 2, 3, 4, or 5. R1 and R1′ are independently alkyl, preferably lower alkyl, and may include one or more isotopes. Suitable R1 and R1′ groups include, for example, —CD3 and —CD2CD3.
In a particular embodiments, a set of diazonium salt analogs are capable of reacting with a naphthol phosphate in a quadruplexed assay to detect and quantify up to four biomarkers, such as an assay for the breast cancer markers ER, PR, Her2, and Ki67. In this example, the diazonium salts increase in molecular weight by increments of 5 daltons.
Although the preceding examples illustrate isotopic labeling with deuterium, other stable isotopes can be used. For example, hydrogen atoms can be replaced by tritium atoms, carbon atoms can be replaced by 13C isotopes, nitrogen atoms can be replaced by 15N isotopes, and/or oxygen atoms can be replaced by 17O isotopes. It also is contemplated that a mixture of isotopes, e.g., 2H and 13C, can be used to create a greater variety of stable-isotope-labeled mass tag precursors.
In some embodiments, isotopically labeled precursors, e.g., aniline-d5, may be commercially available. In other embodiments, mass tags can be synthesized or labeled with isotopes using methods known to those of ordinary skill in the art of organic synthesis. Isotopic labeling can be performed with any mass tag, mass tag precursor, mass tag, mass code, and/or enzyme substrate moiety disclosed herein to produce a series of two or more mass tags, mass tag precursors, mass codes, and/or enzyme substrate moieties suitable for use in multiplexed assays to yield two or more mass codes having the same chemical structure but different molecular weights.
Suitable isotope-labeled mass tags differ in mass sufficiently to produce mass codes that can be distinguished from one another during mass spectrometric detection. A mass spectrometer may be capable of distinguishing mass codes that differ in mass by only 1 dalton. Accordingly, useful isotope-labeled mass tags differ in mass by at least one dalton. In some embodiments, isotope-labeled mass tags differ from one another in mass by at least 1 dalton to at least 50 daltons, more typically at least 1 dalton to about 20 daltons, even more typically at least 1 dalton to about 10 daltons, with working embodiments being exemplified by a mass difference of 1-5 daltons, such as by 1, 2, 3, 4, or 5 daltons. In particular working examples, isotope-labeled mass tags differ from one another in mass by 5 daltons. Due to naturally occurring isotopes, it is expected that any given mass tag, and its corresponding mass code, will have some variability in its molecular weight. For example, some molecules may contain one or more “naturally occurring” deuterium atoms, tritium atoms, 13C atoms, 14C atoms, 15N atoms, etc. Thus, a given mass code may produce MS peaks at the expected m/z value, x, as well as progressively smaller peaks having m/z values of x+n where n is an integer. In particular embodiments, n ranges from 1 to 3 or 1 to 4. Typically, peaks are not seen at x+4 or greater. For example, a reporter ion with an expected m/z of 482 typically will generate progressively smaller peaks at 483, 484, and 485. Although mass codes having a m/z difference of just 1 dalton can be detected separately, if isotope-labeled pairs have a mass difference of less than 4 daltons, there may be at least some overlap between the MS peaks produced by the two mass codes. Any significant overlap can reduce accuracy when quantifying the amount of mass codes corresponding to each target within the tissue sample. A mass difference of at least 4 daltons, or at least 5 daltons, provides insignificant or no overlap between MS peaks produced by the mass codes. Thus, in some embodiments, the mass difference between any given pair of stable-isotope-labeled mass tags, mass tag precursors, or mass codes having the same chemical structure is at least 4 daltons. In particular embodiments, the mass difference is at least 5 daltons.
Particular embodiments concern using nucleic acid sequences or proteins (e.g. Her1, Her2, Her3, Her4, ER, PR, and p95Her2), or combinations of nucleic acids or proteins, as targets in multiplexing analysis, particularly where such detection provides better diagnostic capabilities for a particular malady. Throughout this disclosure when reference is made to a target protein it is understood that the nucleic acid sequences associated with that protein can also be used as a target. In some examples, the target is a protein or nucleic acid molecule from a pathogen, such as a virus, bacteria, or intracellular parasite, such as from a viral genome. As another example, a target protein may be produced from a target nucleic acid sequence associated with (e.g., correlated with, causally implicated in, etc.) a disease.
A target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example a target nucleic acid sequence can have at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues. Similarly, a target polypeptide can vary substantially in size. Without limitation, the target polypeptide will include at least one epitope that binds to a peptide specific antibody, or fragment thereof. In some embodiments that polypeptide can include at least two epitopes that bind to a peptide specific antibody, or fragment thereof.
In specific, non-limiting examples, a target protein is produced by a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) associated with a neoplasm (for example, a cancer). Numerous chromosome abnormalities (including translocations and other rearrangements, amplification or deletion) have been identified in neoplastic cells, especially in cancer cells, such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like. Therefore, in some examples, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., genomic target nucleic acid sequence) amplified or deleted in at least a subset of cells in a sample. In one example, the genomic target nucleic acid sequence is selected to include a gene (e.g., an oncogene) that is reduplicated in one or more malignancies (e.g., a human malignancy). For example, Her2, also known as c-erbB2 or Her2/neu, is a gene that plays a role in the regulation of cell growth (a representative human Her2 genomic sequence is provided at GENBANKTM Accession No. NC_000017, nucleotides 35097919-35138441). The gene codes for a 185 kd transmembrane cell surface receptor that is a member of the tyrosine kinase family. Her2 is amplified in human breast, ovarian, and other cancers. Therefore, a Her2 gene (or a region of chromosome 17 that includes the Her2 gene) can be used as a genomic target nucleic acid sequence, or its protein product can be used as a target protein. Other breast cancer relevant proteins include ER, PR, and Ki67.
Oncogenes are known to be responsible for several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18q11.2 are common among synovial sarcoma soft tissue tumors. The t(18q11.2) translocation can be identified, for example, using probes with different labels: the first probe includes FPC nucleic acid molecules generated from a target nucleic acid sequence that extends distally from the SYT gene, and the second probe includes FPC nucleic acid generated from a target nucleic acid sequence that extends 3′ or proximal to the SYT gene. When probes corresponding to these target nucleic acid sequences (e.g., genomic target nucleic acid sequences) are used in an in situ hybridization procedure, normal cells, which lack a t(18q11.2) in the SYT gene region, exhibit two fusion (generated by the two labels in close proximity) signals, reflecting the two intact copies of SYT. Abnormal cells with a t(18q11.2) exhibit a single fusion signal.
In other examples, a target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is selected that is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), and the pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (that encompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.
The aforementioned examples are provided solely for purpose of illustration and are not intended to be limiting. Numerous other cytogenetic abnormalities that correlate with maladies, such as neoplastic transformation and/or growth are known to those of ordinary skill in the art, or shall be hereafter discovered. Target proteins that are produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have been correlated with neoplastic transformation are useful in the disclosed methods.
In other examples, a target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of the virus- or microorganism-derived target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in a cell or tissue sample is indicative of the presence of the organism. For example, the target peptide, polypeptide or protein can be selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such as Plasmodium falciparum and other Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species). In some examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from a viral genome.
In certain examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV18). In other examples, the target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).
XII. METHOD OF USING DISCLOSED EMBODIMENTS OF THE MASS TAG PRECURSOR CONJUGATEDisclosed embodiments herein concern a method for imaging and/or quantifying one or more targets in a sample, e.g., a tissue sample such as a formalin-fixed, paraffin-embedded tissue sample, using mass tag precursors, mass tags, mass codes, and mass spectrometric (MS) techniques, such as laser desorption/ionization, including laser desorption/ionization (LDI). Some embodiments use an enzyme substrate and a separate mass tag precursor. The enzyme converts the substrate into an active species that can react with the mass tag precursor to deposit a mass tag, such as by precipitation, at the target site. Other embodiments use a conjugate comprising a mass tag precursor coupled directly or indirectly to an enzyme substrate moiety. The mass tag precursor may be bonded to the enzyme substrate moiety by one or more covalent bonds, one or more ionic bonds, any other bonding association sufficiently robust to produce a conjugate suitable for use in the method, and combinations of bonds. The mass tag precursor and the enzyme substrate moiety can be coupled directly together, or can be coupled using a linker.
Other particular embodiments concern using an enzyme substrate and a mass tag precursor conjugate. The enzyme converts the mass tag precursor conjugate into an activated species that is effectively coupled (e.g. covalently bound) to tyrosine moieties proximal to the target, such as at tyrosine moieties on the enzyme, on the specific binding moiety, or on the target.
In certain embodiments concerning samples having multiple targets, each target is identified and detected using a mass tag, typically a different mass tag. For certain disclosed embodiments, the mass tags have the same chemical structure but include different isotopes and/or different amounts of a particular isotope such that each mass tag has a different mass. Using mass tags with the same chemical structure facilitates accurate target quantification, wherein quantification comprises determining the size of a mass peak and correlating it with the amount of the target.
A sample having a target of interest is first contacted with an enzyme, which may be coupled to a specific binding moiety that recognizes a particular target, or region of a target, such as an epitope. The sample is then exposed to the conjugate, or plural such conjugates. A mass tag is deposited at the site of a target in a sample, such as by precipitation, at the target for subsequent detection by MS in the form of a mass code (an ion).
The mass tags as described herein are deposited (covalently or non-covalently) or precipitated at a target location, or multiple target locations, where they are exposed to laser-initiated ionization, producing mass codes, which are detected using LDI mass spectrometry. However, it is contemplated that additional mass spectrometry technologies are adaptable and amenable to the disclosed compositions for tissue target identification. For example, desorption electrospray ionization (DESI; Wiseman et al., 2008, Proc. Natl. Acad. Sci. 105:18120-18125) mass spectrometry, secondary ion mass spectroscopy (SIMS; Boxer et al., 2009, Annu. Rev. Biophys. 38:53-74), laser ablation inductively coupled plasma mass spectrometry (LA-ICP; Becker et al., 2010, Mass. Spectrom. Rev. 29:156-175), and laser ablation flowing atmospheric pressure afterglow ambient pressure mass spectrometry (LA-FAPA; Shelley et al., Anal. Chem. 2008, 80, 8308-8313) are technologies known in the art that could be adapted for use in tissue target identification using the disclosed mass tag precursor conjugate. As such, disclosed embodiments herein are not limited to the method of mass spectrometry utilized for detection purposes.
A general depiction of one embodiment of a method for using deposition of mass tags is provided in
A general depiction of one embodiment of a method for using a mass tag precursor conjugate is provided in
The mass tag precursor, and tyramine or tyramine derivative, or multiple such moieties, also can be used in conjunction with polymers, biomolecules, and/or nanoparticles to achieve deposition of multiple tyramine, tyramine derivatives, and mass tags, thus increasing the chances of reliable detection.
In illustrative embodiments, a multiplexed analysis of a sample is disclosed. One such multiplexed embodiment is illustrated in
Certain disclosed examples of multiplexed analysis include using a first enzyme to produce a first mass tag to detect a first target, and then using a second enzyme to detect a second target. For example, a first target in a sample can be detected using an alkaline phosphatase-catalyzed mass tag deposition approach, as discussed above. A second target can be detected using a second enzyme, such as horseradish peroxidase. A person of ordinary skill in the art will appreciate that even additional targets can be detected using yet a third, or even more, enzymatic reactions to deposit a mass tag at a target in sample. Thus, this detection approach is referred to as a plural enzyme, such as a dual enzyme, system.
Plural enzymes need not be used. Instead, a single enzyme, with plural intervening enzyme deactivation steps can be used. For example, a first target may be detected by a first enzymatic reaction. Residual enzyme from the first aliquot then is deactivated. For example, horseradish peroxidase can be used, and after a first mass tag is deposited at a first target, residual horseradish peroxidase can be deactivated using excess hydrogen peroxide. The second target is then detected using a second mass tag deposited using a second horse radish peroxidase reaction. Particular embodiments of a multiplexing method concern using a mass tag precursor conjugate. One such multiplexed embodiment is illustrated in
Embodiments of the disclosed stable-isotope-labeled mass tag precursors, mass tags, and mass codes are suitable for multiplexed assays, wherein multiple targets in a sample, such as a tissue sample, are imaged and quantified using mass tags and MS techniques. Suitable targets include proteins, peptides, and nucleic acid sequences such as DNA and/or RNA sequences of interest. In some embodiments, the targets are cancer biomarkers. In certain embodiments, an enzyme is immobilized on the target. The enzyme is exposed to a mass tag precursor and an enzyme substrate moiety with which the enzyme is capable of reacting. The reaction forms a mass tag precursor conjugate comprising the enzyme substrate moiety and mass tag precursor, and the mass tag is deposited proximal to the target. A mass code, or reporter ion, is subsequently cleaved from the mass tag and detected by mass spectrometry.
When multiple targets are present, an enzyme is immobilized at each of the targets. Each enzyme is exposed to a mass tag precursor and an enzyme substrate moiety with which the enzyme is capable of reacting, thereby forming mass tags proximal each target. A mass code, or reporter ion, is subsequently cleaved from each mass tag and detected by MS. In some embodiments, the targets are cancer biomarkers. In certain embodiments, the sample is formalin-fixed, paraffin-embedded (FFPE) tissue.
Stable isotope label-based mass spectrometry imaging (SILMSI) has several advantages compared to conventional IHC tissue analysis. In some embodiments, SILMSI provides accurate detection for simultaneous quantification wherein quantification comprises determining the size of a mass peak and correlating it with the amount of multiple cancer biomarkers on FFPE tissue. In certain embodiments, SILMSI techniques demonstrate good linearity (e.g., 0.96≤R2≤0.99) within a dynamic range of 103 in the mass spectrometric peak intensity measurement. SILMSI provides objective, quantitative information for multiple biomarkers, whereas IHC images typically are evaluated subjectively by trained medical personnel. SILMSI can provide a medical practitioner with more accurate information for a patient's cancer diagnosis and prognosis, thereby enabling the practitioner to devise a personalized treatment plan for the patient.
In one embodiment, a first target is contacted with a first enzyme, which may be coupled to a specific binding moiety that recognizes the first target, or a region of the first target, such as an epitope, within the tissue sample. The tissue sample then is exposed to the enzyme substrate moiety and a first mass tag precursor. The enzyme substrate moiety reacts with the enzyme and the mass tag precursor, thereby depositing a mass tag at the site of the target in a sample, such as by precipitation. A subsequent target is contacted with a subsequent enzyme, which may be coupled to a specific binding moiety that recognizes the subsequent target, or a region of the subsequent target. The subsequent enzyme may the same or different, than the first enzyme. The sample then is exposed to the enzyme substrate moiety and a subsequent mass tag precursor. A mass tag is deposited at the site of the subsequent target in the sample. The first mass tag and the subsequent mass tag have substantially the same chemical structure, but differ in molecular weight. The molecular weight is varied by replacing particular atoms in the chemical structure of the mass tag precursor and/or the enzyme substrate moiety with isotopes of those atoms. For example, hydrogen atoms may be replaced with deuterium atoms. The mass codes are subsequently cleaved from the tags and detected by MS. The amounts of the mass codes are quantified to determine the relative amounts of each target in the sample.
With reference to
One embodiment of a method for quantifying plural targets (e.g., cancer biomarkers) on FFPE tissue is shown in
Each mass code produced is detected and the mass code's amounr in the tissue sample section is quantified. The size of the tissue sample section depends, in part, on the laser beam size. For example, a laser beam with a diameter of 30 μm may ionize mass tags in a 30 μm×30 μm section of the tissue sample. The laser beam may have a diameter ranging from a just few microns to several hundred microns. In some embodiments, the laser beam has a diameter in the range of 5-200 μm, 10-100 μm, 15-75 μm or 20-50 μm. For example, the laser beam diameter may be 10 μm, 15 μm, 20 μm, 25 pm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm.
In some embodiments, after mass tags have been ionized in a tissue sample section, the tissue sample and the laser are moved relative to one another. For example, the tissue sample and/or the laser is repositioned such that the laser is capable of ionizing mass tags in a subsequent section of the tissue sample. In certain embodiments, the tissue sample and/or laser is repositioned a plurality of times, and laser ionization/desorption followed by mass code detection and quantification, wherein quantification comprises determining the size of a mass peak and correlating it with the amount of a target, is performed for a plurality of tissue sample sections. In particular embodiments, rastering is performed, i.e., the tissue sample and/or laser is moved in a grid-like pattern such that mass tags in substantially each section of the tissue sample are ionized and the respective mass codes are detected and quantified. For example, if the laser beam has a diameter of 30 μm and the tissue sample size is 0.9 mm×1.2 mm, the tissue sample is “divided” into a grid of 30 ×40 sections and data is obtained for the resulting 1200 sections.
Thus, the mass spectrometric image data obtained correlates to regularly-spaced spatial coordinate points of the tissue sample, and each point may contain mass spectrometric information from a plurality of mass codes. The regularly spaced coordinate points taken together represent an ‘image’ of the sample. To visualize the mass spectra data in two dimensions correlating to the sample sections, relevant information is extracted from the mass spectra at each coordinate and converted to a single scalar value in a two-dimensional array. For example, when detecting two or more targets in a sample, data corresponding to the expected mass codes from each target is extracted from the mass spectra obtained for each tissue sample section (or coordinate) and quantitated. Analytical imaging software can be utilized to convert the data into a two-dimensional image. In a particular embodiment, an in-house program “Ventana Analytical Image” was developed and used.
For each target of interest, a two-dimensional image is created. Each image contains a two-dimensional array of quantitative (numerical) data correlating to the expected mass code produced by a mass tag deposited at a particular target. In some embodiments, the quantitative data may comprise the area, or intensity, of a mass spectrometric peak having a m/z ratio corresponding to an expected m/z ratio of the mass code. In other embodiments, a plurality of mass spectrometric peaks is measured and the measurements combined to quantify the mass code. The plurality of peaks typically includes a primary peak having a m/z ratio of x corresponding to the expected m/z ratio of the mass code and at least one secondary peak having a m/z ratio of x+1, x+2, or x+3.
To create a single image of a tissue sample, numerical data from the individual data arrays corresponding to individual targets is combined by performing mathematical operations on the numerical values to analyze the relationship(s) between values at homologous coordinates. For example, the ratio of two targets, such as ER and PR, may be of interest. In such embodiments, a numerical value corresponding to the ratio of the two targets—as determined by the relative peak sizes produced by the respective mass codes generated from mass tags deposited at each target—at each coordinate is calculated. The numerical values form a single data array representing the target ratios throughout the tissue sample. In theory, the ratio can range from zero to infinity if the mass code corresponding to either target does not produce a measurable peak, such as when there is no mass tag deposited at the target at that particular coordinate. In some embodiments, the ratios range from greater than zero to 100, such as from 0.01 to 100, 0.02 to 50, 0.04 to 25, or 0.1 to 10.
In some embodiments, a visual image representing the numerical data may be desirable. For example, a person reviewing the data may prefer to evaluate the ratios of two targets (e.g., ER and PR) by viewing a color image representing the ratios found in each tissue sample section rather than a numerical grid. In some embodiments, a color or other symbol may be assigned to a range of numerical values, thereby producing a color or symbol map with discrete colors or symbols in a grid-like pattern (see, e.g.,
Most conventional digital image data analysis software is designed to accept positive integer values typically produced by digital cameras. The ability to handle signed decimal fractions and floating point values is typically provided by mathematical array processing tools. In order to visualize floating point values as image data, a custom software interface based on widely available software libraries for scientific data processing and visualization (Python, Numpy, SciPy, VTK, ITK, wxPython) was developed. The software is capable of reading in numerical arrays in floating point format, as text files, and creating color maps in which numerical values may be assigned to different combinations of hue, saturation, value, and alpha. By applying these types of color maps to the numerical arrays resulting from processing of the mass spectrometric data, the data can be visualized and spatial organization of the tissue sample can be correlated with relationships between analytes, in a quantitative manner. The software tool also can gather statistics about data values and render a scale to understand color as a function of the data point value. In some embodiments, the distribution of values and relationship to the color scale (i.e., a color reference guide) is also provided in the same screen alongside the image. In certain embodiments, the range of data values displayed is selected to cover a specified range of interest, e.g., ratios ranging from 0.5-1.5 or 2-4. Narrowing the range displayed may facilitate comparison of data value distribution between samples. The generated heat maps can be saved to provide a convenient record of the data obtained from the sample.
In some embodiments of the disclosed method for detecting plural targets in a tissue sample, the numerical data and/or heat map images produced facilitate diagnosis, prognosis and/or treatment of a disease state. For example, knowing the ratios of cancer biomarkers, such as breast cancer biomarkers, facilitates accurate diagnosis, staging, and/or prognosis of a patient's disease state. In certain embodiments, the numerical data and/or heat map images may be utilized to tailor a treatment plan for a patient. For instance, a patient having a breast cancer tumor with high ratios of ER/PR may benefit from a different treatment plan than a patient whose breast cancer tumor has low ratios of ER/PR.
In some embodiments, the targets may be two or more forms of a particular biomarker, e.g., an intact form and one or more truncated forms. For example, the Her2 biomarker can be found in both intact and truncated forms, e.g., p95Her2 fragments (or Her2 carboxy-terminal fragments). Her2 is a transmembrane receptor with an intracellular domain and an extracellular domain. Metalloproteinases are capable of cleaving proteins like Her2, thereby producing Her2 fragments. In some instances, truncated Her2 is formed when initiation of mRNA translation encoding Her2 begins from different AUG codons within the gene. Several truncated forms of Her2 may form. Approximately 20-40% of Her2-positive tumors express a series of p95Her2 fragments. One truncated form, 110-115 kDa p95Her2, is biologically hyperactive because it easily forms dimers with itself or with Her3, and dimerization leads to breast cancer progression. Another truncated form, 95-100 kDa p95Her2, has a biological activity equivalent to intact Her2. A third truncated form, 90-95 kDa p95Her2, is biologically inactive.
Patients with Her2 positive (as detected by IHC) breast cancer typically are treated with trastuzumab (Herceptin), a monoclonal antibody drug that binds the extracellular domain of Her2 protein. The IHC-based assay for Her2 detection uses a primary antibody (4B5, Ventana Medical Systems, Inc.) capable of recognizing an epitope in the Her2 intracellular domain. In Her2 the extracellular domain is amino acids 1-652, the transmembrane region is amino acids 653-675, and the intracellular domain is amino acids 676-1255. The 4B5 antibody specifically binds to an epitope on the intracellular domain of Her2 including amino acids 1231-1250 of SEQ ID NO: 3 (GAPPSTFKGTPTAENPEYLG).
Hence, the assay cannot distinguish between intact and truncated forms of Her2 and instead detects both forms. A significant disadvantage to this assay, and to the patient's well-being, is that trastuzumab is only effective against the intact form of Her2 since it binds to the extracellular domain. Since truncated Her2 (p95Her2) has no extracellular domain, trastuzumab is therapeutically ineffective in patients with a significant amount of truncated Her2. Another drug, lapatinib, is effective against truncated forms of Her2.
If a tumor includes a significant percentage of truncated Her2, the patient can be treated with lapatinib instead of, or concurrently with, trastuzumab. Lapatinib is a tyrosine kinase inhibitor, which interrupts the Her2 growth receptor pathway.
Currently, lapatinib is used when a patient's cancer continues to progress despite treatment with trastuzumab. Lapatinib also is used when a patient's cancer is “triple positive,” i.e., ER+/EGFR+/Her2+. Thus, an assay that quantifies the intact and truncated forms of Her2 and/or determines a ratio of intact:truncated Her2, can be used to determine the appropriate, targeted treatment for Her2-positive tumors.
Because truncated forms of Her2 lack the extracellular domain, an antibody that binds to the extracellular domain of Her2 will detect only intact Her2 in a tissue sample. Antibodies capable of recognizing and binding to the extracellular domain of Her2 include SP3 (Spring Bioscience) and p95 D9 (Biomatik).
A tissue sample includes primarily truncated Her2 if it is Her2 positive when the 4B5 antibody is used for IHC detection, but Her2 negative when the SP3 or p95 D9 antibody is used. A tissue sample includes primarily intact Her2 if the IHC results are similar with both the 4B5 and SP3 (or p95 D9) antibodies. A tissue sample with a mixture of both intact and truncated forms will have a positive IHC result with the SP3 (or p95 D9) antibody, but will have a more strongly positive result with the 4B5 antibody.
Thus, in some embodiments (see, e.g.,
In certain embodiments, second antibody 168 is capable of recognizing and binding to truncated biomarker 170 and to intact biomarker 154. In such embodiments, second mass tag 180 will be deposited in the vicinity of both truncated biomarker 170 and intact biomarker 154, thereby staining both forms of the biomarker with mass tag 180. Intact biomarker 154 is quantified by quantifying first reporter ion 182. However, quantifying second reporter ion 184 determines the total amount of both intact biomarker 154 and truncated biomarker 170. To quantify truncated biomarker 170 by itself, the peak intensity of first reporter ion 182 is subtracted from the peak intensity of second reporter ion 184.
In a particular embodiment, intact and truncated forms of Her2 are detected and quantified by using SP3 as first antibody 152 and 4B5 as second antibody 168. SP3 binds only to intact Her2, and 4B5 binds to both intact and truncated forms of Her2. In the embodiment shown in
In some embodiments, a visual image representing the numerical data may be desirable. For example, a person reviewing the data may prefer to evaluate the ratios of two targets (e.g., intact Her2 and truncated p95Her2) by viewing a color image representing the ratios found in each tissue sample section rather than a numerical grid. In some embodiments, a color may be assigned to a range of numerical values, thereby producing a color map with discrete colors in a grid-like pattern (similar to
Certain embodiments also can be used for array-based immunoassays. One such array-based immunoassay is illustrated in
A kit for detecting a target in a sample by using mass spectrometry may comprise one or more mass tag precursors selected from the embodiments disclosed herein. In particular embodiments, the mass tag precursor may be conjugated with an enzyme substrate, a tyramine moiety, a tyramine derivative, an optional linker or carrier, and any combination thereof.
A kit for detecting a plurality of targets by mass spectrometry includes at least a first mass tag precursor and a second mass tag precursor. The first and second mass tag precursors have the same chemical structures, and at least one of the first mass tag precursor and the second mass tag precursor is isotopically labeled such that they differ in mass. In certain embodiments, the kit may further include one or more additional mass tag precursors having the same chemical structure as the first and second mass tag precursors. Each additional mass tag precursor is isotopically labeled such that its mass differs from the masses of the first mass tag precursor, the second mass tag precursor, and any other additional mass tag precursor.
In some embodiments, the kit further includes a specific binding moiety-enzyme conjugate, wherein the specific binding moiety is capable of recognizing and binding to a target in a tissue sample or a specific binding moiety bound to a target in a tissue sample. In certain embodiments, the kit includes a plurality of specific binding moiety-enzyme conjugates, wherein each specific binding moiety is capable of recognizing and binding to a particular target in a tissue sample or a specific binding moiety bound to a particular target in a tissue sample. In some embodiments, the specific binding moiety-enzyme conjugate is an antibody-enzyme conjugate. In particular embodiments, the enzyme in each conjugate is the same.
In some embodiments, the kit further includes an enzyme substrate, wherein the enzyme substrate is capable of reacting with an enzyme and a mass tag precursor to produce and deposit a mass tag at a target in a tissue sample. In other embodiments, each mass tag precursor is part of a mass tag precursor conjugate, wherein the enzyme substrate moiety of the mass tag precursor conjugate is capable of reacting with an enzyme to produce and deposit a mass tag at a target in a tissue sample.
In some embodiments, a kit for detecting two targets in a tissue sample includes first and second mass tag precursors, an enzyme substrate, and at least one specific binding moiety-enzyme substrate. In certain embodiments, the enzyme is a phosphatase such as alkaline phosphatase, the enzyme substrate has the chemical structure
and the first and second diazonium salts have the chemical structures
where n is 1-5. In particular embodiments, the first and second diazonium salts have the chemical structures
In some embodiments, a kit for detecting two to four targets in a tissue sample includes a plurality of mass tag precursors, an enzyme substrate, and at least one specific binding moiety-enzyme substrate. In certain embodiments, the enzyme is a phosphatase such as alkaline phosphatase, the enzyme substrate has the chemical structure
and there are two to four mass tag precursors comprising diazonium salts having chemical structures selected from
where n is 0-5, and R1 and R1′ are independently lower alkyl. R1 and R1′ independently are optionally substituted with one or more isotopes. In particular embodiments, the diazonium salts have chemical structures selected from
The following examples are provided to exemplify certain features of working embodiments. A person of ordinary skill in the art will appreciate that the scope of the present invention is not limited to the working features of such examples.
Certain of the following examples concern tissue staining and subsequent mass imaging. A general procedure for tissue staining and mass imaging is as follows:
A formalin fixed paraffin embedded human tissue sample was sectioned at 4 μm and tissue sections were placed on Plus or Superfrost Plus slides (VWR International) and MS tissue sections were placed on indium-tin oxide (ITO) coated glass slides (Bruker Daltonics, Bremen, Germany). Deparaffinization, cell conditioning (antigen retrieval), IHC staining, and/or mass tag application were done using a Benchmark XT or Discovery XT automated IHC instrument (Ventana Medical Systems Inc, Tucson, Ariz., U.S.) and Ventana reagents, unless otherwise specified.
If the mass tag deposited was soluble in alcohol, the sample slides were rinsed with water following completion of the Discovery XT staining protocol, dried for 30 min. at 65° C., and rinsed with hexane before MS analysis.
If the deposited mass tags were insoluble in alcohol, the slides were dehydrated using an ethyl alcohol (80%, 80%, 90%, 90%, 100%, 100%) sequence and dried in air or under vacuum. In general, samples that were exposed under vacuum for an extended period of time provided more intense signals.
A laser desorption ionization (LDI)/time of flight (TOF) linear mass spectrometer using FlexControl acquisition software (Autoflex III, Bruker Daltonics, Bremen, Germany) was used for analysis of the mass tagged tissue sample. A suitable mass range was selected according to the mass of the tags. The mass spectra were collected at each raster point across the designated measurement region. A raster width of 25-100 μm is generally used but can be changed as desired. Unless otherwise specified, the smartbeam laser was operated at a 200 Hz frequency and accumulated 100 shots at each raster point. Laser attenuator and focus settings were chosen to generate a signal with satisfactory intensity and signal to noise ratio. Positive ion mode was used with ion source 1 at 20 kV, ion source 2 at 18.73 kV, the lens at 6 kV, and post ion extraction delay set to 0 ns.
A. In-Situ Hybridization Procedures
In-situ hybridization (ISH) of human tissues in disease states has a well-established place in cancer diagnostics, however there still remains a need for alternatives to ISH technologies that are based on fluorescent and/or chromogenic signal target detection. The embodiments disclosed herein for target detection using Mass Spectrometric Imaging (MSI) detection of genes or gene products provide such alternatives, such as MSI-based multiplexing applications. For example, probes directed to corresponding nucleic acid targets may be labeled with cognate haptens recognized by corresponding anti-hapten antibodies associated with molecular weight tags that are detectable by MSI. Further, fluorescent and chromogenic ISH technologies typically determine staining intensity with categorical values whereas MSI-based ISH allows numeric relative quantification (wherein quantification comprises determining the size of a mass peak and correlating it with the amount) and therefore offers a significant advantage over non-MSI based ISH methods.
In exemplary working embodiments performed to support embodiments of the present invention, silver was utilized as a mass tag precursor for detecting Her2 genomic DNA and human papillomavirus (HPV) genotype targets in mouse xenografts of human cancer cell lines. Exemplary working embodiments were performed utilizing alkaline phosphatase (AP) silver in situ hybridization (SISH) based detection methods as described in U.S. Pat. No. 7,632,652, which we herein incorporate by reference. After hybridizing a nucleic acid probe to the desired target mass tag precursor, deposition and detection, proceeded as previously described for IHC.
B. Indium-Tin Oxide (ITO) Slide Modification Procedures
In particular embodiments, mass tag deposition was carried out utilizing ITO-coated mass spectrometry slides that have been immersed in a gelatin. Effective slide preparation treatments concern preserving the conductive ITO layer on the mass spectrometry slides. Without being limited to a theory of operation, it is currently believed that this prevents any surface charging during the MS ionization process. Particular embodiments concern slides comprising a hydrophilic layer, which promotes mass tag deposition on VMSI' s automated strainers. In particular embodiments, a suitable hydrophilic coating can be obtained by immersing the slides in 1% gelatin (from cold water fish skin, Sigma PN G7041) in 1X PBS for several hours at room temperature, rinsing with distilled water, and air drying the slide at room temperature before tissue placement. This technique was applied to particular working examples using ITO slides.
In other embodiments, the hydrophilic coating comprises a hydrophilic protein coating. Certain embodiments concern immersing the tissue-free slide in a solution of gelatin, such as gelatin from cold water fish skin, a protein, such as bovine serum albumin (BSA), a detergent, such as octylphenoxypolyethoxyethanol (IGEPAL CA-630®), and an organic compound, such as glutaraldehyde to form a cross-linked proteinaceous layer on top of the ITO coating, thus providing a hydrophilic coating to promote automated mass tag deposition without damaging the ITO coating required for mass spectrometric analysis. Particular embodiments concern using a mixture of 0.1% gelatin (Sigma G7041) and 0.1% BSA (Roche), to which was added added 10% IGEPAL CA-630 and 5% glutaraldehyde, followed by thorough mixing. ITO slides (Bruker) were used without pre-cleaning. The protein solution was added onto the non-labeled end of the slide and spread to cover the slide evenly. The solution was allowed to dry in air and placed on a hot plate at 80° C. for about 10 minutes and stored in a slide box. Additionally, applying hydrophilic coatings before tissue placement, such as poly-lysine, amino dextran, and casein, to the ITO coated MS slide can preserve the conductive ITO layer on the slide.
Other treatments for ITO slide preparation were explored; however, these treatments did not sufficiently preserve the conductive ITO layer. Examples of these treatments include cleaning procedures, wherein the slide is immersed in a sequence of different solvents, such as trichloroethylene, acetone, methanol, isopropanol, and potassium hydroxide, and is then air dried. Other cleaning procedures include submerging slides in 20% ethanolamine and sonicating at 80° C., then air drying. Additional methods include hydrophilic coating modifications, such as using Ventana reagents ChipMap Prep 1 (PN 760-4121) and ChipMap Prep 2 (PN 760-4122) during the staining procedure (i.e. after tissue placement). The main components of ChipMap Prep 1 are buffer and detergent, and the main component of ChipMap Prep 2 is casein. An additional approach involved treating the slide with a polyvinylalcohol and glutaraldehyde solution (ChipSpread™) before tissue placement. Other methods include using a combination of cleaning and coating the slides, such as first cleaning the glass slide (tissue sample not yet in place) with 6N HCl, then coating the slide with (3-aminopropyl) triethoxysilane, followed by immersion in 1,4-phenylene diisocyanate.
C. Working Embodiments
Example 1This example concerns using alkaline phosphatase to cleave a phosphate group from a mass tag precursor conjugate, and demonstrates the expected enzymatic reaction and subsequent detection of the mass code (Scheme 23).
To test the substrate, a 1 mg/ml malachite green phosphate solution was made in water. To 200 μl of tris buffer (pH=10) was added 40 μl of the malachite green phosphate solution. The mixture was split into two tubes containing 120 μl each. 10 μl of an alkaline phosphatase (250 μl/ml) solution was added to one tube. Both tubes were then incubated at room temperature. After about 15 minutes the solution comprising alkaline phosphatase became slightly cloudy. The tube without alkaline phosphatase remained clear. Since the hydrolyzed product is not water soluble, the cloudy solution suggested that the alkaline phosphatase catalyzed hydrolysis of the substrate. After allowing the solution to stand overnight, 100 μl of acetonitrile (CH3CN) was added to each tube to dissolve the precipitate, if any. Both sample solutions were further analyzed on MALDI (w/o matrix LDI). The mass code produced by LDI of the sample treated with alkaline phosphatase had a molecular ion peak of m/z=389.22.
Example 2This example demonstrates the use of styryl dyes as mass tag precursors. Styryl dyes generate comparable MS signals to that of a typical triarylmethane dye (triarylmethane dyes are proven mass tags).
LDI results of a mixture of malachite green and 4-[4-(dimethylamino)styryl] pyridine (DMA-SP; Aldrich #394211) at different concentrations produced the following mass codes: DMA-SP=224.573 and malachite green=329.266. The radical cation of DMA-SP was observed, demonstrating the use of styryl dyes as effective mass tags. Additional styryl dyes, such as (E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N,N-dimethylaniline and (E)-2-(4-methoxystyryl)benzo[d]thiazole, demonstrated the same effect for use as mass tags.
Example 3This example illustrates the use of naphthol/triarylmethane diazo mass tag precursors, as described in Scheme 24.
Amino brilliant green (ABG) 1 mg/ml solution was made in 0.01 N hydrochloric acid (HCl) (made from 40 mg/ml ABG stock solution in DMF). NaNO2 solution in water was added to the ABG solution to provide a final concentration of 0.125% sodium nitrate (NaNO2). The color of the solution changed from colorless to light yellow to green in about 0.5 hour. 1-naphthol solution (1 mg/ml) was made in tris buffer (0.1 M, pH=7.5) from a 28 mg/ml dimethylformamide (DMF) stock solution. 50 μl ABG solution was added to 100 μl of 1-naphthol solution. A red precipitate was observed in less than one minute, indicating azo dye formation. The precipitate was separated by centrifugation and the red residue was dissolved in acetonitrile (CH3CN) to give a red solution. LDI of this solution resulted in a peak at m/z at 555.312, demonstrating the presence of the expected mass of the azo-triarylmethane cation.
Example 4Brilliant green (BG), malachite green (MG) and crystal violet (CV) were obtained from Sigma-Aldrich and used without further purification. A 0.3 mM solution of the triarylmethane mass tag precursor (BG or MG or CV) was made in deionized water and stored in a dark dispenser. Formalin fixed paraffin embedded (FFPE) human tonsil tissue was placed on MALDI compatible ITO glass slides (Bruker).
To label a specific epitope on the tissue (Ki-67 or Bcl-2, for example), the deparaffinized tissue was incubated with a primary antibody targeting the epitope (rabbit anti Ki-67 or mouse anti Bcl-2) for a period of time (e.g 16 mins). After washing, the tissue was exposed to a secondary antibody conjugated with alkaline phosphatase (AP) (e.g. ultraView AP multimer from Ventana Medical Systems, Inc.) for a period of time of about 16 to about 32 minutes. After washing, 100 1 of BCIP (Ventana Medical Systems, Inc.) and 100 ul of mass tag precursor solution (0.3 mM of triarylmethane dye) were added to the slide and allowed to incubate on the tissue for 32 minutes. For the control IHC exemplary working embodiment, 100 1 of BCIP and 100 1 of NBT solution (Ventana Medical Systems, Inc. AP Blue Kit) were used to give a dark blue signal indicating the Ki-67 epitope. After incubation, the slides were washed with reaction buffer (Ventana Medical Systems, Inc.). The above steps were all performed automatically in a Ventana Benchmark Discovery staining system.
After automated staining, the ITO slide was washed in water and finally deionized water. The slide was placed in an oven and baked for 20-30 minutes at ˜65° C. to remove most of the water. The slide was rinsed with hexanes and allowed to air dry for at least 10 minutes. The tissue sample labeled with the mass tag (triarylmethane dye) was ready for MS imaging (MSI). Imagine was performed in a
Bruker Autoflex III spectrometer, equipped with a linear TOF detector. No matrix material was applied. A region of interest was selected and a specific scanning step (raster) was selected (usually 30-100 micron).
Azo products formed between a naphthol and a diazonium salt can be used as mass tag precursors for tissue imaging applications. An example is the Ventana AP Red Detection Kit in which naphthol AS-TR phosphate and Fast Red KL are used to generate a red precipitate at the site of the enzyme. The product of the reaction was analyzed under LDI conditions and it was determined that the amide bond in the naphthol was susceptible to laser irradiation. The formation and detection of azo dye-based mass tags, as described in Schemes 3 and 4, is described below. 100 microliters of naphthol AS-TR phosphate was combined with 100 microliters of Fast Red and 100 microliters of alkaline phosphatase multimer as found in Ventana's AP-Red detection kit. A red precipitate formed in 5-10 minutes. The precipitate was collected by centrifugation, and washed with acetonitrile (MeCN). The washed precipitate was isolated by centrifugation, dispersed in acetonitrile (MeCN), cast on a target and analyzed by LDI. The results of this analysis indicated that the major constituent detected was a reaction product of the Fast Red and naphthol.
To demonstrate that the mass tag precursors generated as described above could be used in human tissue, tonsil tissue was stained according to Ventana's AP Red protocol using anti-Ki67 antibody. The stained tissue was imaged under LDI conditions and the specific mass code having a m/z of 347.932 from the cleavage of the naphthol amide bond was observed. Referring now to
According to the method of Example 5, ten additional diazonium salts were tested. See
This example concerns using heteroaryl compounds for forming oligomers detectable by MS under LDI conditions. Initial trials with DAB stained tissue showed detectable signal (DAB dimer) but the background was quite high. Additional compounds were tested by mixing them with HRP and H2O2 and subsequently examining the precipitates on LDI MS. For example, 1,8-DAN gave a peak at m/z=297 and 8-AQ gave peaks at m/z=426 and m/z=567.
Tissue samples were stained with 1,8-DAN and 8-AQ. The initial results indicated 8-AQ gave similar staining as DAB (Ki-67 on tonsil). A tissue sample stained by 8-AQ demonstrated that the oligomers (mostly trimers and tetramers) of 8-AQ can be readily detected on tissue samples without using a matrix (
A frequently used immunohistochemistry (IHC) assay employs alkaline phosphatase conjugated to a secondary antibody which binds a primary antibody to the antigen of interest. The alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP), which tightly binds to the alkaline phosphatase active site, is then oxidized by nitro blue tetrazolium (NBT) to produce a blue precipitate. This assay is modified in a mass tag protocol to use brilliant green (BG) as presumably an oxidizing agent and a detected mass code.
An anti-phospho-4EBP1 (Thr70) rabbit polyclonal primary antibody binds phospho-4EBP1 in formalin-fixed and paraffin-embedded (FFPE) human prostate tissue. A goat-anti-rabbit secondary antibody conjugated to alkaline phosphatase binds the phospho-4EBP1 primary antibody. Following alkaline phosphatase cleavage of the BCIP phosphate group, BCIP is presumably oxidized by BG to generate a blue BCIP dimer precipitate and colorless BG precipitate. The colorless BG precipitate is then ionized by the mass spectrometer laser and detected as a mass code for phospho-4EBP1.
FFPE human prostate tissue was sectioned at 3 μm for IHC and 5 μm for mass spectrometry (MS) analysis. IHC tissue sections were placed on Superfrost Plus slides (VWR International) and MS tissue sections were placed on indium-tin oxide (ITO) coated glass slides (Bruker Daltonics, Bremen, Germany). Deparaffinization, antigen retrieval, IHC staining, and mass tag application were done using a Discovery XT automated IHC instrument (Ventana Medical Systems, Tucson, Ariz., U.S.) and Ventana reagents, unless otherwise specified.
Control and mass tag slides were stained with p4EBP1 (Thr70) primary antibody (Cell Signaling Technology, Beverly, Mass., US), which was diluted 1:5 and incubated on the tissue for 60 min. at 37° C. One IHC control slide was stained with DABMAP (Ventana Medical Systems), a detection kit using a horse-radish peroxidase conjugated secondary antibody, 3,3′-Diaminobenzidine (DAB) substrate and hydrogen peroxide oxidant. A second IHC control slide was stained with an alkaline phosphatase conjugated secondary antibody and detected with BCIP substrate and NBT oxidant. Control slides were counterstained with hematoxylin. The mass tag slide was stained with an alkaline phosphatase conjugated secondary antibody, using BCIP as a substrate and BG (Sigma-Aldrich, St. Louis, Mo., U.S.) as the oxidant and mass tag. Secondary antibodies were incubated 32 minutes at 37° C. before addition of substrates.
Mass tag sample slides were rinsed with water following completion of the Discovery XT staining protocol, dried for 30 min. at 65° C., and rinsed with hexane before MS analysis. No matrix was used.
A laser desorption ionization (LDI)/time of flight (TOF) linear mass spectrometer using FlexControl acquisition software (Autoflex III, Bruker Daltonics, Bremen, Germany) was used for analysis of the mass tagged tissue sample. Mass spectra covering the mass range of 240 Da-1,000 Da were collected at each raster point across the designated measurement region, using a 100 μm raster width. The smartbeam laser was operated at a 100 Hz frequency and accumulated 100 shots at each raster point. Laser attenuator and focus settings were chosen to produce an expected laser diameter of approximately 50 μm. Positive ion mode was used with ion source 1 at 20 kV, ion source 2 at 18.73 kV, the lens at 6 kV, and post ion extraction delay set to 0 ns.Two different cases of human prostate tissue were analyzed for p4EBP1(Thr70) with brilliant green mass tag. Similar staining results were achieved in the HRP/DAB/H2O2 and AP/BCIP/NBT IHC control slides for both tissue cases. Mass tag results in
The resonance stabilized BG carbocation mass code yields an intense signal with LDI mass spectrometry. Low laser powers allow detection of the BG carbocation mass code while minimizing ionization of additional analytes from the tissue, yielding high signal to noise ratios for the desired mass tag. The ability to obtain intense signals without the use of matrix eliminates numerous drawbacks associated with matrix-assisted LDI (MALDI).
MALDI direct imaging analyses were conducted on serial sections of the human prostate cases used for mass tag analysis, using sinapinic acid as matrix. While protein-rich spectra were obtained (data not shown), no signals were detected at the expected m/z ratios for either 4EBP1 or any of its phosphorylated forms. Detection of p4EBP1 using MS analysis has only been possible using the targeted mass tag approach described here. Mass tag distribution is well correlated with p4EBP1 staining as demonstrated in the IHC control slides.
Referring now to
This example concerns the detection of Her2 and HPV DNA in tissue using mass tags. A FFPE multi-block of mouse xenograft tissues containing human breast cancer cell lines of differential Her 2 gene copy number MCF-7 (ATCC HTB-22™; contains 1-2 Her2 gene copies), ZR-751 (ATCC CRL-1500™; contains 2-4 Her2 gene copies), and Calu-3 (ATCC HTB-55™; contains >6 Her2 gene copies) was cut to 4 μm thickness and placed onto an ITO glass slide (Bruker).
The microscope images of AP SISH stained Her2 xenografts, MCF-7, ZR-751, Calu-3, respectively are illustrated in
A Her2 DNA probe was nick-labeled with the hapten 2,4-dinitrophenol (DNP) and incubated on the related tissues for 2hr at 52° C. to target Her2 related genomic DNA (Ventana Medical Systems, 760-4332; 200 ul,). A rabbit anti-DNP primary antibody (Ventana Medical Systems, 780-4335, 100 ul) was incubated on tissues for 20 min. at 37° C. to detect the Her2 DNA probe.
A general schematic diagram for this procedure is illustrated in
An FFPE multi-block of mouse xenograft tissues containing human cervical cancer cell lines of differential HPV genotype copy number C-33 A (ATCC HTB-31™; contains 0 HPV genes), SiHa (ATCC HTB-35™; contains 1-2 HPV genes), HeLa (ATCC CCL-2™; contains 10-50 HPV genes), and CaSki (ATCC CRL-1550™; contains 400-600 HPV genes) was cut to 4 μm thickness and placed onto an ITO glass slide (Bruker).
The microscope images of AP SISH stained HPV xenografts, C-33 A, SiHa, HeLa, and CaSki, respectively, are illustrated in
An HPV DNA probe, nick-labeled with the hapten digoxigenin (DIG) (Ventana Medical Systems, 200 ul of 8 ug/ml), was incubated on the related tissues for 6hrs at 42° C. to target HPV DNA. while a rabbit anti-DIG primary antibody (Sigma-Alrich PN 07782, diluted 1:2,000, 100 ul) was incubated on tissues for 12 min. at 37° C. to detect the HPV DNA probe.
Example 11At this point, the protocol for both Her2 and HPV target detection are the same. As such, regardless of target, following addition of the rabbit anti-hapten antibodies, goat anti-rabbit AP conjugated secondary antibody for recognition of the rabbit primary antibody was added to the slides (100 ul of 20 ug/ml for 32 min. at 37° C.). Rabbit anti-goat gold nanoparticle conjugated antibodies were added (100 ul of 100 nM for 32 min. at 37° C.) and bound to the secondary antibodies, thereby functioning as a seeding site for subsequent silver deposition. Upon addition of AgNO3 (100 ul of 50 nM for 20 min. at 37° C.) and BCIP (Ventana Medical Systems, 95405, 100 ul for 20 min. at 37° C.) to the slides, silver ions were reduced to silver atoms by the alkaline phosphatase resulting in their deposition at the site of the gold nanoparticles. Toning was completed by the addition of AuCl3 (100 ul of 0.20% solution for 4 min. at 37° C.), the signal was amplified with additional AgNO3 (100 ul for 4 min. at 37° C.) and fixed with Na2S2O3 (100 ul for 4 min. at 37° C.). Ag2+ and Ag3+ ions were detected in the MS using conditions described previously.
The above two ISH examples clearly demonstrate the capability of detecting generic materials on FFPE tissue samples using enzymatic amplified mass tag approach, which is not possible using direct or un-amplified MSI method. More importantly, in a suitable dynamic range, the enzymatic mass tag approach could also be established as a relative quantitation method for tissue diagnostics. The microscope images of AP SISH stained HPV xenografts (
Mutiplexing detection of three breast cancer relevant proteins (Her2, ER, PR) has been demonstrated with LDI mass tag detection in FFPE xenografts and human tissue. Mouse xenografts of the following cell lines were used for assay evaluation and development: MCF-7 (breast ductal carcinoma), ZR-75-1 (breast ductal carcinoma), and Calu-3 (non-small cell lung cancer adenocarcinoma). The assay was also demonstrated in human breast tissue samples.
The MCF-7, ZR-75-1, and Calu-3 xenografts were embedded in one paraffin block, so that all three xenografts were placed on one slide. Xenograft and breast tissue blocks were cut at 4 μm and allowed to air-dry onto SuperFrost+ or ITO coated slides. ITO slides were treated with 1% gelatin before tissue placement to facilitate successful mass tag deposition on BenchMark XT.
Staining/mass tag deposition was done on a BenchMark XT using an automated protocol. The general staining scheme used is shown in
AP catalyzes the dephosphorylation of naphthol AS TR phosphate (PN 760-4308); the dephosphorylated product then undergoes an azo coupling with a diazonium salt, producing an insoluble precipitate at the antigen site. The chemistry of the AP catalyzed mass tag deposition is shown below in Scheme 25 (
After deposition of the Her2 mass tag, the primary and secondary antibodies are eluted from the tissue section using 1% SDS (Sigma-Aldrich PN L3771-500) 25 mM Glycine (Bio-Rad PN 161-0718) pH 2 buffer (Pirici, D., Mogoanta, L., et al. (2009) “Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype.” Journal of Histochemistry and Cytochemistry 57(6): 567-575.
ER and PR are then sequentially stained in a similar manner on the same tissue sample. The primary and secondary antibody complex used for PR mass tag deposition is not eluted from the tissue using the glycine/SDS buffer.
Upon completion of the BenchMark XT staining/mass tag deposition, the slides are washed with dilute detergent to remove hydrophobic reagents used on the automated stainer, rinsed with tap water, then dried overnight under high vacuum (˜10−4 torr) before LDI-MS analysis of the intact tissue sample with Bruker Daltonic's Autoflex III mass spectrometer.
Mass spec imaging was done using a laser diameter of approximately 20 μm. Imaging data used a raster width of 30 μm. The mass spec was operated in the positive ion mode with no post-ion extraction delay. Voltages used were: ion source 1 20 kV, ion source 2 18.85 kV, lens 6 kV, and linear detector 1.545 kV. Laser frequency was 200 Hz, and up to 100 shots were summed per raster point. Data was collected in the mass range of 400-500 Da.
Typical data obtained for xenograft samples is shown in
Three different control samples were run for evaluating the xenograft model system: (1) no primary antibody for Her2, ER, or PR; (2) no secondary antibody-alkaline phosphatase conjugate; and (3) no elution of antibodies with glycine/SDS buffer before staining ER and PR. The first two types of control samples showed colorless tissue with no mass tags detected in the mass spectrometer using laser powers equal to that used on samples.
Example 12The exemplary disclosed embodiment described in Example 11 has been expanded to quadruplexed detection of Her2, ER, PR and Ki67 using substantially similar methods to those methods described in Example 11. Quadruplexed detection was demonstrated in a FFPE multiblock containing MCF-7, ZR-75-1, and Calu-3 xenografts. Because Her2 expression in these xenografts is substantially higher than ER, PR or Ki67, a mass tag with considerably lower ionization by LDI-MS was chosen for Her2. This allows mass tag detection for all four antigens using one laser power to sample the MCF-7, ZR-75-1 and Calu-3 xenografts contained on one slide.
In this non-limiting example, the following diazonium salts were used to generate mass tags for each antigen: fast red violet LB (FR V LB, Sigma-Aldrich PN F3381) for Her2; FB BB for ER; FB RR for PR; and FV B for Ki67 (listed in the order of sequential staining). The structure of FR V LB and the detected mass tag are illustrated below. LDI-MS results are shown in
Possible antigen damage caused by the 1% SDS 25 mM glycine pH 2 antibody elution treatment was evaluated for ER, PR, Ki67, and Her2, with the evaluation for each antigen done on separate tissue sections. Xenografts were cut at 4 μm and placed on ITO coated glass slides treated with 1% gelatin. Following antigen retrieval, xenografts were subjected to zero or two cycles of 1% SDS 25 mM glycine pH 2 washing at 50° C. Detection was then completed with the addition of the following: an appropriate primary antibody; AP conjugated secondary antibody; naphthol AS TR phosphate; and FB BB. The FB BB mass tag was detected using LDI-MS. Results for ER are shown in
Possible removal of precipitated diazonium coupling reaction products by the 1% SDS 25 mM glycine pH 2 antibody elution treatment was evaluated. Single antigen detection for ER or Her2 was done on xenograft sections. After all mass tag deposition steps were completed and the mass tag precursor was deposited at the antigen site, slides were treated with zero, one, two, or three rounds of the 1% SDS 25 mM glycine pH 2 antibody elution treatment at 50° C. The intensities of the mass tag obtained from the overall average mass spectrum for the region imaged by LDI-MS are summarized in Table 5. LDI-MS parameters were held constant for the four slides examined for one antigen and one particular mass tag, but were not necessarily held constant for analysis of another mass tag/antigen combination. The SDS glycine buffer treatment did not reduce the intensity of detected mass tags.
Quadruplexed mass tag intensities relative to single antigen mass tag intensities were examined. Quadruplexed detection for Her2, ER, PR and Ki-67 was completed on one slide containing MCF-7, ZR-75-1, and Calu-3 xenografts. Four separate serial section slides were stained individually for each separate antigen, using the same mass tag for each antigen as in the quadruplex slide. Mass tag intensities from the overall average spectrum for the region measured by LDI-MS are summarized in Table 6. The lower intensities observed in the quadruplexed slide relative to the single antigen slides is possibly due to ion suppression, a documented phenomenon in LDI-MS. Without being limited to a particular theory of operation, it is currently believed that the presence of multiple mass tags at the antigen site may lower the intensity of each detected mass tag.
This exemplary embodiment concerns using a small laser diameter for analysis. Laser diameter was estimated to be approximately 50 μm for data shown in Examples 9 and 10. Background silver staining varies heterogeneously across tissue sections, variably affecting the overall intensity measured when ionizing silver atoms via LDI. With a smaller laser diameter, cells showing positive silver staining can be sampled with less overlapping of the laser into negative cells having only background staining, theoretically giving a more accurate measurement of silver intensity in positive cells. Results are shown in
Human breast tissue 4 μm sections were placed on ITO coated glass slides treated with 1% gelatin and stained for Her2 DNA using the AP silver in-situ hybridization procedure described in Example 9. Silver ions were detected with LDI-MS in profiling mode by collecting single spectra at individual raster points. For each raster point sampled, shots were accumulated and summed until silver signal was depleted at one set laser power. The laser power and focus settings produced a diameter of approximately 15 - 25 μm. Exemplary results are shown in
Particular disclosed embodiments concern quantifying enzymatically deposited mass tags. To evaluate the feasibility of quantification (wherein quantification comprises determining the size of a mass peak and correlating it with the amount of a particular target) using LDI-MS detection of enzymatically deposited mass tags, tissue-free exemplary working embodiments were performed in order to eliminate sample variation inherent in tissue samples. ITO coated glass slides were chemically modified to covalently bind protein to the slide without disturbing the ITO coating. Bovine serum albumin (BSA) was spotted on the slide in known concentrations. Staining/mass tag deposition was then completed on the Benchmark XT as described under Example 11, using a mouse anti-BSA primary antibody, and FB BB. The SDS/glycine buffer treatment was not needed in these single antigen exemplary working embodiments; however, a person of ordinary skill in the art would recognize that such a buffer treatment may be used. The FB BB mass tag was detected using LDI-MS.
To allow comparison of LDI-MS results with some orthogonal technique, ImageJ software was used to determine a gray value for each spot before slides were imaged in the MS. Because gray values vary logarithmically with antigen concentration, ImageJ data were fit with a logarithmic trendline. A correlation was observed for spotted BSA concentrations ranging from 0 to 450 ng/mL, which demonstrates the feasibility of quantification (wherein quantification comprises determining the size of a mass peak and correlating it with the amount of a particular target) of LDI-MS detection of enzymatically deposited mass tags. LDI-MS detection of FB BB and ImageJ gray value results are shown in
This particular exemplary embodiment concerns using heteroaryl compounds in conjunction with transition metal ions for enhanced detection by MS under LDI conditions. A particular disclosed embodiment concerns using DAB combined with nickel chloride (NiCl2) for HRP catalyzed staining and subsequent MSI. For this exemplary embodiment, the following general staining procedure was used. Except for the NiCl2 solution, all the other reagents (DAB inhibitor, HRP multimer, DAB chromogen, and DAB H2O2) were obtained from the Ventana ultraView™ Universal DAB detection kit (catalog number: 760-500). After applying the HRP multimer, one drop of DAB chromogen and one drop of NiCl2 (16 mM in water) were applied on the tissue section and incubated for a certain period (e.g. 4-32 minutes). Alternatively, a pre-mixture of DAB chromogen and NiCl2 solution was made separate from the slide and incubated prior to adding onto the tissue. Approximately one drop of DAB H2O2 was then dispensed on the slide and incubated for 8 minutes. After staining, the slides were treated as with standard DAB stain and were further dried under high vacuum. The dried slides were examined in Autoflex III under LDI conditions (e.g. no matrix material used). A representative image is shown in
The staining procedure of this example was based on HRP catalyzed silver (Ag+) reduction in the presence of H2O2 and hydroquinone. General staining methods, as disclosed herein, were used for the disclosed embodiments. For detection, approximately one drop of Silver A reagent was applied and incubated for 4 minutes followed by applying approximately one drop of Silver B and approximately one drop of Silver C and incubation for 8 minutes. After staining, the slides were washed and dehydrated and further dried under high vacuum. The dried slides were examined in Autoflex III under LDI conditions (e.g.
no matrix material used).
Example 17This particular disclosed embodiment concerns using 3-Amino-9-ethylcarbazole (AEC) as an additional example of using heteroaryl compounds as HRP substrate for MSI. General staining methods, as disclosed herein, were used for the disclosed embodiments. AEC chromogen and AEC H2O2 from a Ventana AEC detection kit (Catalog number: 760-020) were applied and incubated for a period of time (e.g. 8-32 minutes) on tissue. After staining, the slide was washed with soapy water and pure water before dried on vacuum. No alcohol dehydration treatment was applied since the precipitation is soluble in alcohol. The dried slide was imaged using Autoflex III under LDI. For the embodiment shown in
A 4-in-1 FFPE multi-cell block of Calu 3, BT-474, SKOV3, and MDA-MB-453 was used to examine whether MSI intensity can be correlated to known protein expression level. The disclosed cell lines have been reported to express different level of Her2 protein, with Calu 3 expressing a higher level of Her2 protein than BT-474, which expresses a level of Her2 protein substantially similar to that of SKOV3, both of which express a higher level of Her2 protein than MDA-MB-453 (e.g. Calu 3>BT-474˜SKOV3>MDA-MB-453). The staining for this particular disclosed embodiment was based on the AP catalyzed naphthol-diazonium reaction. Fast Blue BB (Sigma F3378) was used as the diazonium component, while ultraview Red Naphthol (from Ventana ultraView™ Universal Alkaline Phosphatase Red Detection Kit) was used as the naphthol precursor. Multiple slides were stained using an identical protocol but were imaged at a different point in time in order to examine reproducibility of the intensity data. For each disclosed cell line, multiple regions within a specific cell line section were imaged and
To examine inter-slide variation and reproducibility, the identically stained slides were imaged at different points in time. The average intensity values of the same cell line from different imaging exemplary working embodiments were found to be different. Without being limited to a particular theory of operation, it is currently believed that the difference in average intensity values is likely due to the different instrumental conditions and slightly different imaging set up parameter. However, in particular embodiments, the absolute intensities were normalized against one of the cell line (e.g. Her2 in Calu 3 cell line as 100 AU), with which reproducible relative intensity levels were obtained that correlated with the reported Her2 expression levels (
An FFPE tissue block from MCF-7 nude-mouse xenograft breast cancer (block number: 20718, Ventana Medical System Inc., AZ, USA) was used as the sample for IHC staining and SILMSI analysis. The FFPE tissue block was cut in a microtome to the desired thickness (5 μm) and affixed onto the pretreated indium tin oxide (ITO) coated slide (part number: CG-81IN-S115, Delta Technologies Inc, Minn., USA). The ITO slide was pretreated with gelatin by immersing the ITO slide in gelatin solution 1% w/v gelatin (part number: G7041, Sigma-Aldrich, Mo., USA) in 1x PBS (part number: 14190, Invitrogen, NY, USA) for 5 hours at room temperature. Then, the ITO slide was rinsed with dH2O and allowed to air dry before use.
All immunohistochemistry was performed on Ventana Benchmark XT® automated staining platforms. The tissue samples and primary antibodies were pre-qualified (i.e., assayed for suitability and performance) using ultraView Universal Alkaline Phosphatase Red Detection Kit (part number:760-501, Ventana Medical System Inc., AZ, USA). In all cases one drop of reagent is equal to 100 L.
FFPE MCF-7 nude-mouse xenograft breast cancer tissue mounted on Superfrost Plus Micro Slide (part number: 48311, VWR, Ill., USA) was stained by ultraView Universal Alkaline Phosphatase Red Detection Kit (part number:760-501, Ventana Medical System Inc., Ariz., USA) for ER detection in one slide and PR detection in another slide. The same tissue section from the same tissue block was used for the IHC staining. Optical images of the IHC stained slides were taken with light microscopy (Axio Imager A2, Carl Zeiss MicroImaging Inc., Gottingen, Germany).
FFPE MCF-7 nude-mouse xenograft breast cancer tissue mounted on a gelatin-pretreated ITO slide was stained by multiplex staining protocol using in-house synthesized stable-isotope-labeled chromogens. The same slide was sequentially stained for ER detection and PR detection. There were two independent IHC staining exemplary working embodiments for LDI MS imaging analysis. In one IHC staining exemplary working embodiment (Experiment-1), IHC staining was performed using light form of chromogen for ER detection and heavy form of stable-isotope-labeled chromogen for PR detection in the one ITO slide. In another IHC staining exemplary working embodiment (Experiment-2), IHC staining was performed using light form of chromogen for PR detection and heavy form of stable-isotope-labeled chromogen for ER detection in the another ITO slide. The two IHC stained ITO slides were analyzed by LDI MS imaging analysis, separately.
Example 19Referring now to Scheme 16, the following was synthesized.
Stable-Isotope-Labeled (SIL) Mass Tag Synthesis and FormulationCompound (1a)—Light benzene diazonium salt: According to Scheme 26, aniline 0.3 mL (3.3 mmol, 1 equivalent, Sigma 10400) was dissolved in 1.3 ml of d.i. H2O. Tetrafluoroboric acid 1.02 mL (5.6 mmol, 1.7 equivalents, Sigma 207934) was added dropwise and the mixture subsequently chilled to 0° C. Sodium nitrite 228 mg (3.3 mmol, 1 equivalent, Fluka 71759) was dissolved in 0.9 mL of d.i. H2O and added dropwise to the reaction. The reaction was stirred for 30 minutes. The precipitate was filtered, washed with d.i. H2O. The residue was dissolved in a minimum volume of acetone and precipitated with cold diethyl ether. The precipitate was filtered, washed with cold diethyl ether and dried under vacuum. An off-white powder was isolated in 60% yield. 1H NMR (400 MHz, CDCl3) δ (CDCl3) 8.00 (2.0H, dd, J=7.92, 8.56 Hz), 8.30 (1.0H, t, J=7.72 Hz), 8.64 (1.9H, d,
J=7.84 Hz). 13C NMR (101 MHz, CD3OD) δ 141.2, 132.3, 131.3. IR (cm−1) 3105 (C—H), 2292 (N≡N), 1565, 1458, 1307, 1283, 1026, 751, 662.
Compound (1b)—Heavy benzene diazonium salt: According to Scheme 26, d5-Aniline 0.3 mL (3.1 mmol, 1 equivalent, Sigma 175692) was dissolved in 1.3 ml of d.i. H2O. Tetrafluoroboric acid 1.02 mL (5.6 mmol, 1.7 equivalents, Sigma 207934) was added dropwise and the mixture subsequently chilled to 0° C. Sodium nitrite 215 mg (3.1 mmol, 1 equivalent, Fluka 71759) was dissolved in 0.9 mL of d.i. H2O and added dropwise to the reaction. The reaction was stirred for 30 minutes. The precipitate was filtered, washed with d.i. H2O. The residue was dissolved in a minimum volume of acetone and precipitated with cold diethyl ether. The precipitate was filtered, washed with cold diethyl ether and dried under vacuum. An off-white powder was isolated in 51% yield. IR (cm−1) 3342 (C-D), 2361, 2292 (N≡N), 1524, 1303, 1017.
Compounds 1a and 1b were each formulated in 10 mM acetate buffer to a final concentration of 5 mM.
Example 20Multiplex Staining Protocol—ER Light/PR Heavy (Experiment 1)
Multiplex Staining Protocol—PR Light/ER Heavy (Experiment 2)
The experiment above was repeated with PR being stained with compound la and ER being stained with compound 1b to determine whether there was a quantifiable difference when labeling a given antigen with a light or heavy mass tags. The expected reporter ions and spectra are illustrated in
LDI MS Imaging Analysis
LDI-TOF MS measurements were done on an autoflex III™ MALDI-TOF mass spectrometer equipped with a smartbeam laser' (Bruker Daltonics Inc, Bremen, Germany). The sample was measured with the LDI-TOF MS (linear mode) controlled with flexControl software (version 3.3) and flexImaging software (version 2.1). The laser diameter was set to “small” with a 30 μm of laser-diameter and 30 μm of imaging resolution. Laser frequency was 200 Hz with 100-shot per data acquisition. The laser power was set to 40%, and the mass range of data acquisition was 60˜600 Da. The other instrument parameters were set as followings: Ion Source 1 (20 kV), Ion Source 2 (18.85 kV), Lens (6 kV), Detector Electronic Gain (Enhanced), Detector Gain Voltage Offset (linear, 1,300 V). Moving the laser in increments of 30 μm across the tissue sample, 1,444 spectra were obtained from Experiment 1 and 1,177 spectra were obtained from Experiment 2.
The chromogens used in the IHC staining, i.e., the light form of chromogen (compound 1a) and the heavy stable-isotope-labeled chromogen (compound 1b), have the exact chemical structure and composition. The only difference is the replacement of hydrogen in light form of chromogen with deuterium in heavy stable-isotope-labeled chromogen. Thus, the efficiency of IHC chemical reaction should be exactly the same in the two independent experiments (see
Moreover, the IHC chemical reaction generated azo-dye precipitates (
Acquired LDI MS imaging measurements were exported from flexImaging to Excel (Microsoft Office 2003, SP3, Microsoft Corporation, Wash., USA) for quantitative analysis. Data from each measurement is composed of the peak intensity and its corresponding coordinates for ER and PR, separately. The relative quantization (i.e., the ratio of ER/PR) was calculated by the comparison of the peak intensity of ER versus PR in two independent experiments. The quantitative results were saved as a .txt file and exported to in-house developed software for imaging analysis (Ventana Analytical Image, Ventana Medical System Inc., Ariz., USA).
FFPE MCF-7 nude-mouse xenograft breast cancer tissue mounted on Superfrost Plus micro slide was stained by ultraView Universal Alkaline Phosphatase Red Detection Kit for ER detection in one slide and PR detection in another slide. The optical images of the IHC stained slides were taken with light microscopy. As shown in
Quantitative results of the ER/PR analysis by the SILMSI technique from Experiments 1 and 2 were processed by comparing the peak intensity of the reporter-ion for ER and the reporter-ion for PR . Ten representative data points from Experiment lare shown in Table 8.
Table 9 summarizes the results and compares the two experiments using four categories of ER/PR ratio, i.e., ER/PR <0.67, ER/PR E (0.67, 1.50), ER/PR E (1.50, 4.0), and ER/PR ≥4.00. Each “MS event” is one MS spectrum obtained as the laser was rastered across the tissue sample in 30 μm increments.
By analyzing the quantitative results from the two independent experiments, it is clear that the ER expression level is higher than that of PR in MCF-7 nude-mouse xenograft breast cancer tissue. In the category of ER/PR E (1.50, 4.00), there are 44.17% of measurements in Experiment-1 and 45.88% of measurement in Experiment-2. Interestingly, in the category of ER/PR E (0.67, 1.50), there are 51.18% of measurements in Experiment-1 and 51.23% of measurement in Experiment-2. These results indicate that about 45% of measurements show up-regulated ER compared to PR expression in the MCF-7 nude-mouse xenograft breast cancer tissue, and about 50% of measurements show that ER and PR have similar expression levels. Only 4.22% of measurements from Experiment-1 show ER/PR <0.67, and 2.72% of measurements from Experiment-2 show ER/PR <0.67.
To investigate the influence of stable-isotope-label order (direction) on the quantitative analysis of ER/PR by SILMSI, two independent experiments were performed as described above to demonstrate that the results were not affected based on which antigen was labeled with the light mass tag and which antigen was labeled with the heavy mass tag.
To investigate the reliability, or reproducibility, of the SILMSI technique, the results from Experiments 1 and 2 were statistically analyzed (
To investigate the dynamic range and linearity of the SILMSI technique, the peak intensities for ER (
The ER/PR ratio of each 30×30 μm section of the tissue samples from Experiments 1 and 2 was processed in Microsoft Excel®, and “heat-maps” were generated using three different cutoffs of ER/PR. When ER/PR ≤0.67, the corresponding section on the heat map was colored green. When ER/PR ≥1.50, the corresponding section was colored red. When ER/PR ∈ (0.67, 1.50), the corresponding section was colored yellow. The number in each box is the numerical ratio of ER/PR determined from the actual measurement on the tissue section. The tissue sample from experiment 1 measured 1.14 mm×1.14 mm, and the tissue sample from Experiment 2 measured 1.05 mm×0.96 mm.
LDI-TOF MS measurements are performed. Multiple spectra are obtained by moving the laser in increments across the tissue sample. Imaging measurements are exported for quantitative analysis. Data for each measurement is composed of the peak intensity and its corresponding coordinates for the light mass code (intact Her2 only) and heavy mass code (truncated and intact Her2). For each measurement, the relative quantization (i.e., the ratio of intact Her2/total Her2) is calculated by comparison of the peak intensity of the light mass code versus the heavy mass code. The ratio of intact Her2/truncated Her2 is calculated by comparing the peak intensity of the light mass code to the difference between the peak intensities of the heavy and light mass codes (i.e., heavy mass code peak intensity—light mass code peak intensity).
General Procedures for Tissue Staining and Mass Imaging using Exemplary Embodiments of the Disclosed Mass Tag Precursor Conjugate: A formalin fixed paraffin embedded (FFPE) tissue sample can be stained on a Ventana Benchmark XT instrument with standard reagents. After deparaffinization and suitable cell conditioning (antigen retrieval), the specific target of interest in the tissue sample is labeled with an antibody-HRP conjugate to localize peroxidase on the target. The tissue is exposed to a mixture of 100 L of H2O2 from a Ventana UltraView™ Detection Kit and 100 L of tyramine or tyramine derivative substrate solution at a suitable concentration, usually in the range of 1 to 200 M. The reaction is allowed to proceed at 37° C. for a certain period of time (e.g., 4-60 minutes). After the reaction, the slide is washed thoroughly with buffers and water. The slide can be dried under vacuum or in an oven to remove water. Matrix material such as CHCA, SA or DHB is applied on the tissue using a Bruker Imageprep sprayer. The matrix-coated sample is imaged using an AutoFlex III MALDI spectrometer (Bruker). Reference is made to
With reference to Scheme 18, a nitrophenyl photo-cleavable linker 166 (Advanced ChemTech, RT1095) was first loaded onto a trityl chloride resin 164. The Fmoc protective group of protected amine 168 was removed in 2% 1,8-diazabicyclo[5.4.0]undec-7-ene/dimethylformamide (DBU/DMF) for 10 minutes to provide amine 170. An Fmoc-protected amino acid was activated using standard peptide coupling reagents diisopropylcarbodiimide (DIC)/ hydroxybenzotriazole (HOBt) and conjugated to the amino group of the nitrophenyl linker on resin 172. After the coupling reaction, the Fmoc group was subsequently removed in 2% DBU/DMF. The final conjugate 174 was cleaved from the resin in a suitable trifluoroacetic acid (TFA) cocktail to afford a nitrophenyl conjugate 176 with a free carboxylic acid group for further conjugation with tyramine.
Example 23With reference to Scheme 19, tyramine was conjugated to the nitrophenyl photo-cleavable linker carboxylic acid 176 through a standard carbodiimide-mediated coupling reaction. To one equivalent of 1 in DMF was added 1.5 eq of DCC and NHS to afford the active ester 178. The NHS ester 178 was used without separation in the next reaction by adding 2 to 5 eq of tyramine. After stirring at room temperature for at least 4 hours, the insoluble material was removed by filtration and the solution was concentrated. The tyramide conjugate 180 was further purified by HPLC. This method is used to produce a variety of detectable mass codes, having, for example the following m/z ratios.
A tyramide conjugate, CDO-PEG8-Arg-PL-Tyr (illustrated below) was synthesized according to the general methods described in Example 1 and 2. The molecule was cleaved under MALDI conditions to give a detectable ion at m/z=840.
The tyramide tag was deposited at tissue target (Ki-67 on tonsil tissue) through HRP mediated oxidative addition reaction using the general tissue staining procedure and scheme in
Another exemplary working embodiment was performed on a Her2 3-in-1 xenograft slide with anti-Her2 (Ventana 4B5) as the primary antibody. A substantially similar procedure to that of Example 3 was followed.
The following U.S. Patent, patent publications, and applications are assigned to Ventana Medical Systems, Inc., the assignee of the present application, and each is incorporated herein by reference: U.S. Pat. No. 7,695,929; U.S. Patent Publication No. 2007/0117153; U.S. Patent Publication No. 2006/0246524; U.S. Patent Publication No. 2006/0246423; U.S. patent application Ser. No. 12/154,472; U.S. Provisional Application No. 61/328,494; U.S. Provisional Application No. 61/398,946; and U.S. Provisional Application No. 61/464,216.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
Claims
1. (canceled)
2. A conjugate comprising having a formula:
- wherein the ‘Mass Tag Precursor’ includes a metal chelate; R16 is selected from ether, hydroxyl, or —(nitrogen—R11)—, R11 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group; R17 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group; n is an integer ranging from 1 to 20; Y is selected from oxygen, sulfur, or —(nitrogen—R11)—; the optional linker is selected from an aliphatic group, a heteroaliphatic group, or heterobifunctional linkers;
- and the optional carrier is selected from a polymer, a biomolecule, a liposome, a micelle, or a nanoparticle.
3. The conjugate of claim 2, wherein the metal chelate mass tag precursor is a metal-heteroaryl chelate having a formula:
- where L is selected from nitrile, thiocyanate, hydroxyl, thiol, amine, carbonyl, phosphonyl, halide, phosphine, or a heteroaliphatic group; Y and Z are independently selected from oxygen, sulfur, and —(nitrogen—R11)—; M is a metal selected from Zn, Ni, Co, Ru, Cd, Pt, or Fe; and R11 is an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, or hydrogen.
4. The conjugate of claim 3, wherein the metal-heteroaryl chelate has the formula:
- where R29, R30, R31, R32, and R33 independently an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, a carbonyl group, a halogen, a hydrogen, a hydroxyl group, an isothiocyanate group, an isocyanate group, a nitrile group, a nitro group, a thiol, or any combination thereof; M is a metal selected from Zn, Ni, Co, Ru, Cd, Pt, or Fe; rings A, A′, B, B′, C, C′ are optionally bonded to provide a monoheteroaryl chelate, a biheteroaryl chelate, or a terheteroaryl chelate; Y is selected from oxygen, sulfur, or —(nitrogen—R11)—; and Z is selected from oxygen, sulfur, or—(nitrogen—R11)—, where R11 is an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, or hydrogen.
5. The conjugate of claim 4, wherein the metal-heteroaryl chelate has one of the formulas:
6. The conjugate of claim 5, wherein the conjugate has any one of the formulas:
7. The conjugate of claim 2, wherein the conjugate has a formula:
- where two Amino Acids form a chelate with M, the two amino acids optionally being a peptide or in a peptide chain, and wherein R8 is a linker, a charged species, a chromophore, or any combination thereof; R16 is selected from ether, hydroxyl, and —(nitrogen—R11)—, where R11 is an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, or hydrogen; R17 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group; n is an integer ranging from 1 to 20; and Y and Z are selected from oxygen, sulfur, or —(nitrogen—R11)—.
8. The conjugate of claim 7, wherein the metal is Ni.
9. The conjugate of claim 8, wherein the two amino acids are both histidine.
10. The conjugate of claim 2, wherein
- is tyramide or a tyramide derivative.
11. A conjugate comprising having a formula:
- wherein the ‘Mass Tag Precursor’ includes a nitrophenyl moiety or a heteroaryl moiety; R16 is selected from ether, hydroxyl, or —(nitrogen—R11)—, R11 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group; R17 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group; n is an integer ranging from 1 to 20; Y is selected from oxygen, sulfur, or —(nitrogen—R11)—; the optional linker is selected from an aliphatic group, a heteroaliphatic group, or heterobifunctional linkers; and the optional carrier is selected from a polymer, a biomolecule, a liposome, a micelle, or a nanoparticle.
12. The conjugate of claim 11, wherein the nitrophenyl moiety has the formula:
- where R22, R23, and R24 are independently selected from an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, a carbonyl group, a halogen, a hydrogen, a hydroxyl group, an isothiocyanate group, an isocyanate group, a nitrile group, a nitro group, a thiol, or any combination thereof, at least one of R22, R23, and R24 is nitro; Y is selected from oxygen, sulfur, or —(nitrogen—R11)—; R7 is selected from aliphatic or heteroaliphatic; and R8 is selected from an aliphatic group, a heteroaliphatic group, a heterobifunctional linker, a charged moiety, a chromophore, or any combination thereof; and R11 is an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group.
13. The conjugate of claim 12, wherein the linker has the following general formula where each X is independently selected from CH2, oxygen, sulfur, or —(nitrogen—R11)—; R18 is carbonyl or sulfoxyl; n is an integer ranging from 1 to 20; and p is 0 or 1.
14. The conjugate of claim 12, wherein the nitrophenyl moiety has any of the formulas:
15. The conjugate of claim 11, wherein the heteroaryl moiety has a formula:
- where R25, R26, R27, and R28 are independently selected from carbon or nitrogen, where at least one of R25, R26, R27, and R28 is nitrogen; Y is selected from oxygen, sulfur, or —(nitrogen—R11)—; and R8 comprises an aliphatic group, a heteroaliphatic group, a heterobifunctional linker, a charged moiety, a chromophore, or any combination thereof; and R11 is an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group.
16. The conjugate of claim 15, wherein the linker has the following general formula
- where each X is independently selected from CH2, oxygen, sulfur, or —(nitrogen—R11)—; R18 is carbonyl or sulfoxyl; n is an integer ranging from 1 to 20; and p is 0 or 1.
17. The conjugate of claim 15, wherein the heteroaryl moiety has a formula:
18. The conjugate of claim 11, wherein
- is tyramide or a tyramide derivative.
19. A conjugate comprising having a formula:
- wherein the ‘Mass Tag Precursor’ includes a triarylmethane moiety or a naphthol-azo moiety, R16 is selected from ether, hydroxyl, and —(nitrogen—R11)—, R11 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group; R17 is selected from an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group, n is an integer ranging from 1 to 20, Y is selected from oxygen, sulfur, or —(nitrogen—R11)—; the optional linker is selected from an aliphatic group, a heteroaliphatic group, or heterobifunctional linkers; and the optional carrier is selected from a polymer, a biomolecule, a liposome, a micelle, or a nanoparticle.
20. The conjugate of claim 19, wherein
- is tyramide or a tyramide derivative.
21. The conjugate of claim 19, wherein the ‘Mass Tag Precursor’ has a formula:
- where R2, R3, R4 are selected from an aliphatic moiety or an electron-donating heteroatom-containing moiety, wherein the electron-donating heteroatom-containing moiety is selected from ether (RaORb), hydroxyl (RaOH), silyl ether (RaRbRcSiORd), phosphine (PRaRbRc), thiol (RaSH), thioether/sulfide (RaSRb), disulfide (RaSSRb), isothiocyanate (RaNCS), isocyanate (RaNCO), amine (NH2, NHRa NRaRb)amide (RaNRbC(O)Rc), ester (RaOC(O)Rb), wherein Ra represents one or more of the aryl rings of the triarylmethane; and Rb, Rc, and Rd are independently hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, or any combination thereof; and R19 is selected from oxygen, sulfur, and —(nitrogen—R11)—; and where R11 is an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, or hydrogen.
Type: Application
Filed: Aug 4, 2018
Publication Date: Jan 3, 2019
Patent Grant number: 10883999
Inventors: Rui Hong (Oro Valley, AZ), Hong Wang (Oro Valley, AZ), Mark Lefever (Oro Valley, AZ), Jan Froehlich (Oro Valley, AZ), Christopher Bieniarz (Tucson, AZ), Brian Daniel Kelly (Tucson, AZ), Phillip Miller (Tucson, AZ)
Application Number: 16/055,046
